RNA nanoparticles for brain tumor treatment

ABSTRACT

The presently-disclosed subject matter relates to an artificial RNA nanostructure molecule and method to treat brain tumor in a subject. More particularly, the presently disclosed subject matter relates to a RNA nanostructure containing a multiple branched RNA nanoparticle, a brain tumor targeting module, and an effective amount of a therapeutic agent. Further, the presently disclosed subject matter relates to a method of using the RNA nanostructure composition to treat brain tumor in a subject having or at risk of having brain tumor.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 15/554,360, which issued as U.S. Pat. No. 10,584,144 on Mar. 10,2020, which is a § 371 National Stage Application of PCT/US2016/021447filed Mar. 9, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/130,459, filed Mar. 9, 2015, the entire disclosuresof which are hereby incorporated by reference in their entireties.

GOVERNMENT INTEREST

This invention was made with government support under CA151648 (P.G.),EB012135 (P.G.), CA152758 (C.M.C.), CA175875 (I.N.), CA163205 (I.N.),P30NS045758 (B.K.), R01064607 (B.K.), R01CA150153 (B.K.), andP01CA163205 (B.K.) awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 30, 2018, isnamed 2935720-7_SL.txt and is 4,791 bytes in size.

TECHNICAL FIELD

The presently-disclosed subject matter relates to an artificial RNAnanostructure molecule and method to treat brain tumor in a subject.More particularly, the presently disclosed subject matter relates to aRNA nanostructure containing a multiple branched RNA nanoparticle, abrain tumor targeting module, and an effective amount of a therapeuticagent. Further, the presently disclosed subject matter relates to amethod of using the RNA nanostructure composition to treat brain tumorin a subject having or at risk of having brain tumor.

INTRODUCTION

The most common primary brain tumors in adults are glioblastomas, whichare also one of the most deadly cancers (1). For glioblastomas,conventional treatment involves surgical resection followed by radiationand concurrent chemotherapy. Even with this treatment regimen, themedian survival of patients with glioblastoma is less than 15 months.The poor prognosis is primarily due to tumor recurrence, which isthought to originate from a subset of cancer stem cells that survive theprimary treatments. Recent studies suggested that glioblastoma stem cellsurvived the therapeutic stresses and become more aggressive when theyrecur, developing resistance to the primary chemotherapy.

Bacterial virus phi29 DNA packaging RNA (pRNA) molecule is a crucialcomponent in the phi29 DNA packaging motor and contains two functionaldomains. The intermolecular interaction domain is located at the centralregion (bases 23-97) and within this domain there are two loops (righthand loop and left hand loop) which are responsible for the hand-in-handinteraction through the four complementary base sequences within thesetwo loops. The other domain is a DNA translocation domain which islocated at the 5′/3′ paired ends. The right hand loop (bases 45-48) andthe left hand loop (bases 82-85) allow for the formation of pRNA dimers,trimers and hexamer rings via intermolecular base-pairing via theinteraction of two interlocking loops, the pRNA molecules form dimers,trimers, hexamers, and patterned superstructures [7]. This property offorming self-assembled nanostructure makes pRNA ideal building blocksfor bottom-up assembly. RNA nanotechnology has been rapidly growing as anew generation platform for biological and medical application (2-3). Asnanotechnology rapidly evolves, many attempts have been made to deliversmall interfering RNA (siRNA) using viruses, liposome, lipid, andpolymer based nanoparticles (4).

Clearly there remains a need for improved composition and methodstargeting both brain tumor cells and glioblastoma stem cells to treatthe primary brain tumor and prevent tumor recurrence is desired. Thepresently disclosed subject matter relates to RNA nanoparticlecontaining compositions useful for prophylactic and therapeutictreatment for brain tumors.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. This Summary does notlist or suggest all possible combinations of such features.

In some embodiments, the presently disclosed subject matter provides anartificial RNA nanostructure molecule. The molecule includes a multiplebranched RNA junction motif comprising at least one RNA oligonucleotide,and a brain tumor targeting module, and the module is coupled to an RNAjunction motif. In some embodiments, the molecule further includes atleast one bioactive agent coupled to the RNA junction motif. Anon-limiting example of the bioactive agent is a therapeutic agent. Insome embodiments, the RNA oligonucleotide is at least 6 nucleotides inlength. In some embodiments, the RNA oligonucleotide includes at leastone chemical modification at the 2′ position. Non-limiting examples ofthe chemical modification include 2′Fluoro, 2′ Amine, 2′O-Methyl, or acombination thereof.

In some embodiments, the multiple branched RNA includes a nucleotidesequence 5′-UUG CCA UGU GUA UGU GGG AUC CCG CGG CCA UGG CGG CCG GGA G-3′(SEQ ID NO: 6). In some embodiments, the multiple branched RNA includesa sequence 5′-GATAAGCT CTC CCG GCC GCC ATG GCC GCG GGA T-3′ (SEQ ID NO:7). In some embodiments, the multiple branched RNA junction motif is athree-branched RNA junction motif. In some embodiments, thethree-branched RNA junction motif includes a packaging RNA (pRNA)three-way junction (3WJ) motif. In some embodiments of the presentdisclosure, the RNA molecules form dimers, trimers, hexamers, andpatterned superstructures.

In some embodiments, the presently disclosed subject matter providesthat a branch of the three-branched RNA junction motif includes an a3WJRNA module. In some embodiments, a branch of the three-branched RNAjunction motif includes a b3WJ RNA module. In some embodiments, a branchof the three-branched RNA junction motif includes a c3WJ RNA module. Insome embodiments, the three-branched RNA junction motif includes an a3WJRNA module, a b3WJ RNA module, and a c3WJ RNA module. A non-limitingexample of RNA module include nucleotide sequences 5′-UUG CCA UGU GUAUGU GGG-3′ (SEQ ID NO: 1), 5′-CCC ACA UAC UUU GUU GAUCC-3′ (SEQ ID NO:2), and 5′-GGA UCA AUC AUG GCA A-3′ (SEQ ID NO: 3).

In some embodiments, the diameter of the molecule is at least about 40nm or less. In some embodiments, the diameter of the molecule is atleast about 30 nm or less. In some embodiments, the diameter of themolecule is at least about 15 nm or less.

In some embodiments, the RNA molecule has a zeta potential ranging fromabout −50 mV to about 50 mV. In some embodiments, the molecule has azeta potential ranging from about −25 my to about 25 mV.

In some embodiments, the presently disclosed subject matter providesthat the brain tumor targeting module in the artificial RNAnanostructure molecule includes a ligand that binds to at least onebrain tumor cell surface marker. Non-limiting examples of the braintumor surface marker includes folate receptor, EGFR, transferrinreceptor, and an RGD. In some embodiments, the ligand includes anaptamer. In some embodiments, the aptamer binds to EGFR, PDGFR, folatereceptor, or a combination thereof. In some embodiments, In someembodiments, the targeting module is a folate.

In some embodiments, the presently disclosed subject matter provides abioactive agent includes a drug, a fluorescent dye, a chemical, or acombination thereof. In some embodiments, the bioactive agent includes asiRNA, a miRNA, an anti-miRNA, a ribozyme RNA, an antisense RNA, or acombination thereof. In some embodiments, the bioactive agent isdirected to a brain tumor marker. Non-limiting examples of the bioactiveagent include siRNA sequence and microRNA sequence. In some embodiments,the microRNA molecule is at least 3 nucleotide in length. In someembodiments, the bioactive agent is an anti-miRNA molecule for a miRNAencoding miR-9, miR-10b, miR-21, miR-17, or miR-26. In some embodiments,the bioactive agent is a miRNA molecule for a miRNA encoding let-7a,miR-10b, miR-25, miR-34a, miR-124, miR-145, or miR-181b. In someembodiments, the miRNA includes miRNA locked nucleic acid (LNA)molecule. In some embodiments, the microRNA sequence is an anti-miR-21sequence. In some embodiments, non-limiting examples of the miRNAsequence comprises 5′-GATAAGCT-3′,5′-AGCACTTT-3′, or 5′-ATTTGCAC-3′. Insome embodiments, the miRNA includes an miRNA locked nucleic acid (LNA)molecule. In some embodiments, the bioactive agent includes a LNA miRNAmolecule 5′-+G+A+T+A+A+G+C+T-3′. In some embodiments, miRNA LNA moleculeincludes a sequence 5′-+A+G+C+A+C+T+T+T-3′. In some embodiments, miRNALNA molecule includes a sequence 5′-+A+T+T+T+G+C+A+C-3′.

In some embodiments, the microRNA is a locked nucleic acid (LNA)sequence. In some embodiments, the microRNA is a LNA-miR21 sequence5′-+G+A+T+A+A+G+C+T-3′. In some embodiments, the siRNA binds to a mRNAsequence of a gene that promotes tumorigenesis, angiogenesis, cellproliferation, or a combination thereof, in the brain or spinal cord. Insome embodiments, the siRNA binds to a mRNA molecule that encodes aprotein including pro-tumorigenic pathway proteins, pro-angiogenesispathway proteins, pro-cell proliferation pathway proteins,anti-apoptotic pathway proteins, or a combination thereof. In furtherembodiments, the mRNA molecule encodes a protein including but notlimited to VEGF pathway proteins, EGFR pathway proteins, MGMT pathwayproteins, Rafl pathway proteins, MMP pathway proteins, mTOR pathwayproteins, TGFβ pathway proteins, or Cox-2 pathway proteins, or acombination thereof. In some embodiments, non-limiting examples ofprotein include VEGF, EGFR, POK, AKT, AGT, RAF, RAS, MAPK, ERK, MGMT,MMP-2, MMP-9, PDGF, PDGFR, IGF-I, HGF, mTOR, Cox-2 and TGFβ1. In someembodiments, the siRNa binds to a mRNA molecule that encodes RAS, cMET,HER2, MDM2, PIK3CA, AKT, CDK4, or a combination thereof.

Further provided, in some embodiments of the presently disclosed subjectmatter, is a nucleic acid composition. The composition includes atherapeutically effective amount of the artificial RNA nanostructuremolecule as disclosed above. In some embodiments, the compositionincludes a pharmaceutically acceptable carrier.

Still further, the presently disclosed subject matter, in someembodiments, provides a nanoparticle delivery system. The deliverysystem includes the artificial RNA nanostructure molecule as disclosedabove. In some embodiments, the nanoparticle delivery system furtherincludes a pharmaceutically acceptable carrier.

In another aspect, the presently disclosed subject matter provides, insome embodiments, a method of treating a brain tumor in a subject havingor at risk of developing a brain tumor. The method includesadministering to the subject a therapeutically effective amount of acomposition comprising an artificial RNA nanostructure molecule asdisclosed herein. In some embodiments, the composition includes apharmaceutically acceptable carrier. In some embodiments, the subject isa mammal or a non-mammal vertebrate. In some embodiments, the subject isa human. In some embodiments, the brain tumor is glioblastoma.

Further, in some embodiments, the present disclosure provides a methodof preventing brain tumor recurrence a subject having or at risk ofhaving brain tumor recurrence. The method includes administering to thesubject a therapeutically effective amount of a composition comprisingan artificial RNA nanostructure molecule as disclosed herein. In someembodiments, the composition includes a pharmaceutically acceptablecarrier. In some embodiments, the subject is a mammal or a non-mammalvertebrate. In some embodiments, the subject is a human. In someembodiments, the brain tumor is glioblastoma.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the presently disclosed subject matter are set forthwith particularity in the appended claims. A better understanding of thefeatures and advantages of the presently disclosed subject matter willbe obtained by reference to the following detailed description that setsforth illustrative embodiments, in which the principles of the subjectmatter are used, and the accompanying drawings of which. The drawingswere originally published in color, incorporated by reference in theirentireties (Tae Jin Lee, et al. (2015) Oncotarget, Vol. 6, No. 17,14766-14776). The black and white drawings of the instant applicationcorrespond to the color ones published.

FIG. 1, panels A-D are diagrams and images illustrating construction andcharacterization of multi-functional pRNA-3WJ RNP for glioblastoma celltargeting. A, Construction map of trivalent FA-Alexa647-pRNA-3WJ-si(Luc)RNP harboring three functionalities to form: Folate (FA) as a targetingligand; Alexa647 as an imaging module; and luciferase siRNA for genesilencing. FIG. 1A discloses SEQ ID NOS 19, 3, 2 and 5, respectively, inorder of appearance. B, Atomic force microscopy (AFM) image showingthree-branched triangular structure of self-assembled trivalentFA-pRNA-3WJ-si(Luc) RNP. C, Dynamic light scattering (DLS) data showingthe size of FA-pRNA-3WJ-si(Luc) RNP. D, Zeta potential ofFA-pRNA-3WJ-si(Luc) RNP. The data in C and D were obtained from threeindependent experiments.

FIG. 2, panels A-D are graphs and images showing FA-mediated humanglioblastoma cell targeting by FA-Alexa647-pRNA-3WJ RNP in vitro and invivo. A, Flow cytometry analysis for FA-dependent human glioblastomacell U87EGFRvIII targeting in vitro by FA-Alexa647-pRNA-3WJ RNP.Alexa647 signals from U87EGFRvIII cells treated with 200 nM ofFA-Alexa647-pRNA-3WJ RNP were compared to control RNP (FA-freeAlexa647-pRNA-3WJ RNP) normalized to PBS control. Percentage of cellpopulations were analyzed by student t-test (p<0.001, n=4). B,Immunofluorescence confocal microscopy for FA-dependent humanglioblastoma cell U87EGFRvIII targeting in vitro by FA-Alexa647-pRNA-3WJRNP (middle) in comparison to control RNP (FA-free Alexa647-pRNA-3WJ)(top) or 1 mM free folate pre-treated cells in culture media (bottom).Pseudocolor was used for nucleus (blue), cytoskeleton (green) andAlexa647 (red). C, U87EGFRvIII-induced brain tumors in mice targeted byFA-Alexa647-pRNA-3WJ RNP. Tumors were determined by MM (yellow arrows intop panel) and visualized by fluorescence in vivo imaging (bottom panel)after tail vein injection of FA or FA-free Alexa647-pRNA-3WJ RNP.Representative images from each group of 4 were displayed. D, ANOVAanalysis on fluorescence intensity of Alexa647 normalized by tumorvolume (mm³), p=0.019 (n=4).

FIG. 3, panels A-E are graphs and images showing gene silencing effectof FA-pRNA-3WJ-si(Luc) RNP in human glioblastoma cells and derivedtumor. A, A wide range (up to 400 nM) of FA-pRNA-3WJ-si(Luc) (closedcircles) or FA-pRNA-3WJ-si(Scrm) (negative control, open circles) RNPswere incubated with U87EGFRvIII-Luc cells in vitro (n=4). The change ofluciferase activity was monitored versus the concentration of the RNPs.B, Luciferase gene silencing effect of FA-pRNA-3WJ-si(Luc) in vivo aftertotal of three injections. Luciferase activity change byFA-pRNA-3WJ-si(Luc) (closed circles) or FA-pRNA-3WJ-si(Scrm) (opencircles) were compared by mean bioluminescence intensity (n=5), p=0.007.C, Representative in vivo MRI images for tumor volume andbioluminescence intensity for luciferase activity from bothFA-pRNA-3WJ-siRNA(Luc) or FA-pRNA-3WJ-si(Scrm) after three injections.D, Tumor volumes calculated from MRI compared to scrambled control groupat day 13 post-xenograft, p=0.468 (n=5). E, Mean fluorescence intensitydivided by tumor volume (mm³) was used to normalize the variation amongthe tested mice, p=0.015 (n=5). All error bars indicate s.e.m., andstudent t-test was used for statistical analysis.

FIG. 4, panels A-C are graphs and images showing FA-mediated targetingof human glioblastoma patient-derived stem cell and derived brain tumorby FA-Alexa647-pRNA-3WJ RNPs in animal trials and biodistribution study.A. Flow cytometry analysis for in vitro targeting of human glioblastomapatient-derived stem cell, 1123, by FA-Alexa647-pRNA-3WJ orAlexa647-pRNA-3WJ RNP co-treated with CD44-FITC antibody. PBS andCD44-FITC treated cells were used as gating controls. B, Mouse braintumor derived from 1123 cells was evaluated by MRI for tumor sizedetermination (top). After systemic administration ofFA-Alexa647-pRNA-3WJ RNP, FA-dependent targeting was visualized byfluorescence in vivo imaging in comparison to FA-free Alexa647-pRNA-3WJRNP. C, Biodistribution profile of FA-Alexa647-pRNA-3WJ RNP was obtainedby imaging fluorescence against Alexa647 from major internal organscollected together with brain.

FIG. 5 is graphs illustrating flow cytometry analysis for FA-mediatedhuman glioblastoma cell targeting by FA-Alexa647-pRNA-3WJ RNP in vitro.Human glioblastoma cells U87EGFRvIII were pre-treated with 1 mM freefolate in culture media for 1 hr before incubation withFA-Alexa647-pRNA-3WJ RNP containing medium. Events as a function ofAlexa647 signal intensity detected from U87EGFRvIII cells treated with200 nM of FA-Alexa647-pRNA-3WJ RNP were compared to control RNP (FA-freeAlexa647-pRNA-3WJ RNP). The figure is representative of threeexperiments.

FIG. 6 contains images showing FA-mediated human glioblastoma cell T98Gin vitro targeting by FA-Alexa647-pRNA-3WJ RNPs. Glioblastoma multiforme(GBM) cell line, T98G, was treated with 200 nM of FA-Alexa647-pRNA-3WJor Alexa647-pRNA-3WJ for 1 hr, followed by fluorescence confocalmicroscopy. Pseudocolor was used for nuclear (blue), cytoskeleton(green) and Alexa647 (red).

FIG. 7 includes images showing confocal fluorescence imaging of frozensectioned brain tumor derived from human glioblastoma patient-derivedstem cell 1123 demonstrating the distribution and accumulation ofFA-Alexa647-pRNA-3WJ RNP in brain tumor cells (yellow arrows).Pseudocolor was used for nuclear (blue), and Alexa647 (red).

FIG. 8 includes images showing human glioblastoma patient-derived stemcell 1123-derived mouse brain tumor targeting by FA-Alexa647-pRNA-3WJRNPs with RNA dose-dependent (100>20 μg/mouse) manner. The fluorescenceintensity for the tumor-bearing mouse brains were evaluated at 15 hrsafter systemic injection of RNPs.

FIG. 9A is a diagram illustrating the construction map of trivalentFA-3WJ-LNA-miR21. Panel A discloses SEQ ID NOS 6, 3, 2 and 7,respectively, in order of appearance. FIG. 9B includes graphsillustrating FA-mediated in vitro human glioblastoma cell targetingdetermined by flow cytometry.

FIG. 10 includes images showing FR-dependent human glioblastoma celltargeting visualized by confocal fluorescent microscopy.

FIG. 11 includes images showing confocal fluorescent microscopy analysisvisualizes human glioblastoma cell specific distribution.

FIG. 12 includes graphs and images showing anti-tumor effect ofsystemically delivered FA-3WJ-LNA-miR21 RNP in human glioblastoma cellsderived tumor in vivo.

FIG. 13 is a graph illustrating knock-down of endogenous miR-21 in mousetumor by systemically delivered FA-3WJ-LNA-miR21.

FIG. 14, panels A and B are graphs and images showing down regulation ofmiR-21 by systemically delivered FA-3WJ-LNA-miR21 induced apoptoticpathway through recovery of Pten protein expression.

FIG. 15 is a graph showing knock-down of endogenous miR-21 in mousetumor by systemically delivered FA-3WJ-LNA-miR21 improved overallsurvival of brain tumor-bearing mice.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom.

In certain instances, nucleotides and polypeptides disclosed herein areincluded in publicly-available databases, such as GENBANK® andSWISSPROT. Information including sequences and other information relatedto such nucleotides and polypeptides included in such publicly-availabledatabases are expressly incorporated by reference. Unless otherwiseindicated or apparent the references to such publicly-availabledatabases are references to the most recent version of the database asof the filing date of this Application.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod. As used herein, ranges can be expressed as from “about” oneparticular value, and/or to “about” another particular value. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. For example, if the value “10” isdisclosed, then “about 10” is also disclosed. It is also understood thateach unit between two particular units are also disclosed. For example,if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The presently-disclosed subject matter relates to RNA nanostructuremolecule and method to treat brain tumor in a subject. Moreparticularly, the presently disclosed subject matter relates to amolecule containing a multiple branched RNA junctive motif, a braintumor targeting module. Further, the presently disclosed subject matterrelates to a method of using the RNA nanostructure composition to treatbrain tumor in a subject having or at risk of having brain tumor.

In some embodiments, the presently disclosed subject matter provides anartificial RNA nanostructure molecule. The molecule includes a multiplebranched RNA junction motif comprising at least one RNA oligonucleotide,and a brain tumor targeting module, and the module is coupled to an RNAjunction motif. In some embodiments, the molecule further includes atleast one bioactive agent coupled to the RNA junction motif. In someembodiments, the RNA oligonucleotide is at least about 6 nucleotides inlength.

RNA nanotechnology has recently emerged as an important field due torecent finding of its high thermodynamic stability, favorable anddistinctive in vivo attributes (US 2014/0179758, hereby incorporate byreference in its entirety). In some embodiments of the presentdisclosure, as disclosed in US2014/0179758, the RNA molecules formdimers, trimers, hexamers, and patterned superstructures. Further, RNAnanoparticles can be fabricated with precise control of shape, size andstoichiometry, as demonstrated by the packaging RNA (pRNA) of thebacteriophage phi29 DNA packaging motor, which forms dimmers, trimers,and hexamers via hand-in-hand interactions of the interlocking loops.

In some embodiments, the presently disclosed subject matter relates to aRNA nanoparticle based composition. Such nanoparticles is deliveredsystemically and specifically target intracranial tumors with minimaltoxicity. In some embodiments, the nanoparticle relates to a pRNAthree-way junction (pRNA-3WJ). The pRNA-3WJ of the bacteriophage phi29DNA packaging motor can be used to fabricate a RNA nanoparticle (RNP)with precise control of shape, size and stoichiometry (4-10). Creationof boiling resistant RNPs with controllable shapes and definedstoichiometry has been recently reported (11).

The term “RNA” refers to a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a β-D-ribofuranose moiety. Theterms encompass double stranded RNA, single stranded RNA, RNAs with bothdouble stranded and single stranded regions, isolated RNA such aspartially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA, or analog RNA, thatdiffers from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of an siRNA or internally, for example at one or morenucleotides of the RNA. Nucleotides in the RNA molecules of thepresently disclosed subject matter can also comprise non-standardnucleotides, such as non-naturally occurring nucleotides or chemicallysynthesized nucleotides or deoxynucleotides. These altered RNAs can bereferred to as analogs or analogs of a naturally occurring RNA.

In some embodiments, the RNA oligonucleotide of the RNA nanoparticlesincludes at least one chemical modification at the 2′ position.Non-limiting examples of the chemical modification include 2′Fluoro, 2′Amine, 2′O-Methyl, or a combination thereof. In one embodiments, thepRNA-3WJ nanoparticles with 2′-Fluoro (2′-F) modifications of U and Cnucleotides renders the RNPs resistant to RNase degradation enhancingtheir in vivo half-life while retaining authentic functions of theincorporated modules (7, 12, 13). Furthermore, the pRNA-3WJ RNPs werenon-toxic, non-immunogenic, and displayed favorable biodistribution andpharmacokinetic profiles in mice (14). These favorable characteristicsmake this novel platform attractive for the systemic delivery of siRNAto glioblastoma. One promising ligand for nanoparticle therapy inglioblastoma targeting is folate, a natural member of the B-vitaminfamily. Folate is required for early neuronal development anddifferentiation (15). Its transportation across the blood-cerebrospinalfluid barrier (BCSF) occurs by the choroid plexus (16). The choroidplexus expresses the largest amount of folate receptor (FR) in a body,while no FR expression is detected in cerebellum, cerebrum or spinalcord (17, 18).

In some embodiments, the multiple branched RNA includes a nucleotidesequence 5′-UUG CCA UGU GUA UGU GGG AUC CCG CGG CCA UGG CGG CCG GGA G-3′(SEQ ID NO: 6). In some embodiments, the multiple branched RNA includesa sequence 5′-GATAAGCT CTC CCG GCC GCC ATG GCC GCG GGA T-3′ (SEQ ID NO:7). In some embodiments, the presently disclosed subject matter providesthat a branch of the three-branched RNA junction motif includes an a3WJRNA module. In some embodiments, a branch of the three-branched RNAjunction motif includes a b3WJ RNA module. In some embodiments, a branchof the three-branched RNA junction motif includes a c3WJ RNA module. Insome embodiments, the three-branched RNA junction motif includes an a3WJRNA module, a b3WJ RNA module, and a c3WJ RNA module. A non-limitingexample of RNA module include nucleotide sequences 5′-UUG CCA UGU GUAUGU GGG-3′ (SEQ ID NO: 1), 5′-CCC ACA UAC UUU GUU GAUCC-3′ (SEQ ID NO:2), and 5′-GGA UCA AUC AUG GCA A-3′ (SEQ ID NO: 3).

In some embodiments, the diameter of the molecule is at least about 40nm or less. The diameter is at least about 35 nm or less, at least about30 nm or less, at least about 25 nm or less, at least 20 nm or less, atleast 15 nm or less, at least 10 nm or less, at least 5 nm or less.

In some embodiments, the molecule has zeta potential ranging from about−150 mV to about 150 mV. The RNA molecule has a zeta potential rangingfrom about −140 mV to about 140 mV, from about −130 mV to about 130 mV,from about −120 mV to about 120 mV, from about −110 mV to about 110 mV,from about −100 mV to about 100 mV, from about −90 to about 90 mV, formabout −80 mV to about 80 mV, from about −70 mV to about 70 mV, fromabout −60 mV to about 60 mV. In some embodiments, the RNA molecule has azeta potential ranging from about −50 mV to about 50 mV. The moleculehas a zeta potential ranging from about −45 my to about 45 mV, fromabout −40 mV to about 40 mV, from about −35 mV to about 35 mV, fromabout −35 mV to about 30 mV, from about −35 mV to about 20 mV, fromabout −25 mV to about 15 mV.

In some embodiments, the RNA nanostructure molecule is substantiallystable in pH ranges from about 2 to about 13. The RNA molecule issubstantially stable in pH about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and13. As used herein, the term “substantially stable” can refer tophysical and/or chemical stability. As will be recognized by those ofordinary skill in the art, the term “substantially stable” can refer tostability of the composition under certain conditions, relative to aninitial composition (i.e., when a particular batch of the composition isinitially prepared). In this regard, as will be recognized by those ofordinary skill in the art, one manner in which stability of a particularembodiment of the composition can be determined is as follows: preparinga batch of the embodiment of the composition, making an initialassessment of a sample of the composition (control sample), subjecting asample of the composition to conditions of interest (e.g., storage at aparticular temperature for a particular time period) (test sample),making an assessment of the test sample, and comparing the assessment ofthe control sample to the assessment of the test sample. Calculationscan be made to determine whether the amounts present in the test sampleare 100%±20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, 1, 0.5, or 0.1% of the amount that is in the control sample.

In some embodiments, the presently disclosed subject matter providesthat the brain tumor targeting module in the artificial RNAnanostructure molecule includes a ligand that binds to at least onebrain tumor cell surface marker. As used herein, cell surface markersinclude any cellular component that may be detected within or on thesurface of a cell, or a macromolecuie bound or aggregated to the surfaceof the cell. As such, cell surface markers are not limited to markersphysically on the surface of a cell. For example, cell surface markersmay include, but are not limited to surface antigens, transmembranereceptors or coreceptors, macromolecules bound to the surface, such asbound or aggregated proteins or carbohydrates, internal cellularcomponents, and the like. Non-limiting examples of the brain tumorsurface marker includes folate receptor, EGFR, transferrin receptor, andan RGD. In some embodiments, the ligand includes an aptamer. In someembodiments, the aptamers binds against EGFR, PDGFR, or folate receptor.In some embodiments, the targeting module is a folate.

In some embodiments, a brain tumor targeting module is coupled to theRNA nanoparticle. The targeting module direct the nanoparticle to thebrain tumor cells, to enhance binding to them, to enhanceinternalization, to enhance targeting to cellular enzymes, DNA, RNA,proteins, lipids, or carbohydrates. Non-limiting examples of the braintumor targeting module are antibodies, antibody fragments, polypeptides,cell ligands, aptamers, DNA, RNA, drugs, compounds that enhancetargeting the brain tumor cell, and other groups or materials thatenhance binding to brain tumor cells.

In some embodiments, a brain tumor targeting module may be an antibody.The antibody may have an ability to recognize and specifically bind to atarget on tumor cells and tissues. Non-limiting example of the antibodyis an antibody configured to specifically bind a protein selected frombut not limited to EGFR, human epidermal growth factor (HER), laminin411, insulin-like growth factor (IGF) and tumor necrosis factor-alpha(INF-a).

In some embodiments, a targeting module is an antibody of a classdescribed as antagonist antibodies, which specifically bind to a braintumor stem cell marker protein and interfere with, for example, ligandbinding, receptor dimerization, expression of a brain tumor stem cellmarker protein, and/or downstream signaling of a cancer stem cell markerprotein. Yet in other embodiments, a targeting module is an antibody ofa class described as agonist antibodies which specifically bind to abrain tumor stem cell marker protein and promote, for example, ligandbinding, receptor dimerization, and/or signaling by a cancer stem cellmarker protein. Yet in further embodiments, a targeting module is anantibody that does not interfere with or promote the biological activityof a brain tumor stem cell marker protein and may instead function toinhibit tumor growth by, for example, antibody internalization and/orrecognition by the immune system.

In some embodiments, the targeting module may include a lectin oranother ligand specific to the transferrin receptor. A brain tumortargeting module may further e a ligand to one of any number of cellsurface receptors or antigens, such as RGD.

Further examples of the targeting module is a chemical molecule, a smalldrug molecule or a chromophore molecule, or a protein molecule, or alectin that are covalently joined to polymaleic acid in constructing theconjugation with the RNA nanoparticle.

The term “folate” as used herein can comprise, for example, a genus ofwell-defined B-vitamin compounds, including but not limited to,5-methyltetrahydro folate, 5-formyltetrahydrofolate, dihydrofolate,tetrahydrofolate, folic acid and other folate compounds. Since folate isan essential component required during DNA replication and methylationin highly proliferating cells, many cancer cells, such as those of thebrain, ovary, lung, breast, kidney, endometrium, colon and bone marrow,over-express FRs to increase folate uptake (19). Folic acid (FA), asynthetic oxidized form of folate, has been widely used as a ligand invarious cancer targeting materials (20).

In some embodiments, the presently disclosed subject matter provides abioactive agent includes a drug, a fluorescent dye, a chemical, or acombination thereof. In some embodiments, the bioactive agent includesan imaging module. Non-limiting examples of the imaging module isfluorescent dye, including a non-limiting example Alexa647. In someembodiments, the bioactive agent is coupled to the RNA nanostructuremolecule. In some embodiments, the bioactive agent is a therapeuticagent. In some embodiments, the bioactive agent includes a siRNA, amiRNA, an anti-miRNA, a ribozyme RNA, an antisense RNA, or a combinationthereof. In some embodiments, the bioactive agent is directed to a braintumor marker. Non-limiting examples of the bioactive agent include siRNAsequence and microRNA sequence.

RNA interference (RNAi) is a polynucleotide sequence-specific,post-transcriptional gene silencing mechanism effected bydouble-stranded RNA that results in degradation of a specific messengerRNA (mRNA), thereby reducing the expression of a desired targetpolypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164;U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp,Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001);Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated bydouble-stranded polynucleotides as also described hereinbelow, forexample, double-stranded RNA (dsRNA), having sequences that correspondto exonic sequences encoding portions of the polypeptides for whichexpression is compromised.

The terms “small interfering RNA”, “short interfering RNA”, “smallhairpin RNA”, “siRNA”, and shRNA are used interchangeably and refer toany nucleic acid molecule capable of mediating RNA interference (RNAi)or gene silencing. See e.g., Bass, Nature 411:428-429, 2001; Elbashir etal., Nature 411:494-498, 2001a; and PCT International Publication Nos.WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO99/07409, and WO 00/44914. In one embodiment, the siRNA comprises adouble stranded polynucleotide molecule comprising complementary senseand antisense regions, wherein the antisense region comprises a sequencecomplementary to a region of a target nucleic acid molecule (forexample, a nucleic acid molecule encoding BRCAA1). In anotherembodiment, the siRNA comprises a single stranded polynucleotide havingself-complementary sense and antisense regions, wherein the antisenseregion comprises a sequence complementary to a region of a targetnucleic acid molecule. In another embodiment, the siRNA comprises asingle stranded polynucleotide having one or more loop structures and astem comprising self complementary sense and antisense regions, whereinthe antisense region comprises a sequence complementary to a region of atarget nucleic acid molecule, and wherein the polynucleotide can beprocessed either in vivo or in vitro to generate an active siRNA capableof mediating RNAi. As used herein, siRNA molecules need not be limitedto those molecules containing only RNA, but further encompass chemicallymodified nucleotides and non-nucleotides.

In some embodiments, the siRNA molecule of the presently disclosedsubject matter is a siRNA molecule that binds to a single stranded RNAmolecule, which is a messenger RNA (mRNA) that encodes at least part ofa peptide or protein whose activity promotes tumorigenesis,angiogenesis, or cell proliferation in the brain or spinal cord of amammal, or which is a microRNA (miRNA) whose activity promotestumorigenesis, angiogenesis, or cell proliferation in the brain orspinal cord of a mammal. In some embodiment of the present disclosure,to interfere oncogenic coding genes to regress brain tumor growth, theRNA nanostructure molecule silence oncogenes, including but not limitedto, RAS, cMET, HER2, MDM2, PIK3CA, AKT, and CDK4.

The phrase “brain tumor marker” as used herein refers to genes or geneproducts (e.g., RNA molecules or proteins) which are characteristic ofsome or all of the cells in brain cancer. A brain cancer marker withdiagnostic value can be a gene or gene product expressed in normal,non-cancerous cells, but is characteristic of a type or classificationof cancer by, for example, its over-expression or under-expression ascompared to its expression in normal, non-cancerous cells. A brain tumormarker with prognostic value is a gene or gene product for which theover-expression or under-expression confers predictive information aboutthe future aggressiveness of a cancer and/or its response to therapy atthe time of diagnosis. In a cancer sample, the patterns of expression ofdiagnostic and prognostic cancer markers allow one to accuratelyidentify and determine the future course of the disease, respectively.Non-limiting examples of brain tumor biomarkers are described inWO2007069882 (herein incorporated by reference in its entirety).

In one embodiment, the siRNA molecule binds to an mRNA that encodes atleast part of a peptide or protein whose activity promotestumorigenesis, angiogenesis, or cell proliferation, or a combinationthereof, in the brain or spinal cord of a mammal. Such may be the casewhen the mRNA molecule encodes a protein in a pro-tumorigenic pathway,pro-angiogenesis pathway, pro-cell proliferation pathway, oranti-apoptotic pathway. For example, the protein can be a VEGF pathwayprotein, EGFR pathway protein, MGMT pathway protein, RAF pathwayprotein, MMP pathway protein, mTOR pathway protein, TGFβ pathwayprotein, or Cox-2 pathway protein. In one embodiment, the protein is oneof the following, including but not limited to, VEGF, EGFR, PDK, AKT,AGT, RAFl, RAS, MAPK, ERK, MGMT, MMP-9, PDGF, PDGFR, IGF-I, HGF mTOR,Cox-2, or TGFβ1. In another embodiment, the protein is VEGF, EGFR, MGMT,MMP-2, MMP-9, or PDGF. In still another embodiment, the protein is RAFI,mTOR, Cox-2, or TGFβ1.

In some embodiments, the present disclosure provides that the bioactiveagent is a microRNA sequence. As used herein, the term “MicroRNAs(miRNAs)” as used herein are single-stranded, or double strandednon-coding RNAs, at least about 6 nucleotide in length that can regulategene expression at the post-transcriptional level by either degradingtheir target mRNAs or inhibiting their translation (See, e.g. Bartel, D.P., (2004), Cell, 116,281-297; Liang Z., et al., (2013), J Genet.Genomics, 40, 141-142). MiRNAs play important roles in regulating cellcycle, proliferation, differentiation, metabolism, and apoptosis. Acompendium of microRNA and respective microRNA binding sequences isavailable at the miRNA registry. (See, e.g., Griffiths-Jones et al.(2006) Nucl. Acids Res. 34:D140-D144, US20140045709, herein incorporateby reference in their entireties.) In particular embodiments, themicroRNA and microRNA binding sequence employed in the presentdisclosure are associated with a disease or condition, wherein anantagonist or agonist to the microRNA would be useful in preventing ortreating the disease or condition. Dysregulation of miRNAs has beenimplicated in tumor initiation, progression, and metastasis in severalcancer types (See, Carlin G. A., et al., Nat. Rev. Cancer 2006, 6,857-866; Di L. G., et al., Annu. Rev. Pathol. 2014, 9, 287-314; Garzon,R. et al., Annu. Rev. Med. 2009, 60, 167-179; Iorio, M. V., et al.,Cancer Res 2005, 65, 7065-7070; Croce, C. M., et al., Cell 2005, 122,6-7.). MiRNAs hold great potentials for cancer therapy particularlybecause one miRNA can regulate a broad set of target genes efficientlyand simultaneously, and can therefore address the heterogeneous natureof cancer. Naturally occurring miRNA further displays reduced immuneresponse and low toxicity. Both anti-miRNAs to knockdown oncogenicmiRNAs and mimics of miRNAs to upregulate endogenous miRNAs have beendeveloped as therapeutic strategies to achieve tumor regression (Henry,J., et al. Pharm Res 2011, 28, 3030-3042). However, the major limitingfactor is the ability to specifically deliver these therapeutic modulesto affected cells and tissues. Nanotechnology holds great promise inthis regard and several nanoplatforms have been pursued, but effectivestrategies to inhibit tumor progression are still lacking (Grodzinski,P.; Torchilin, V.; (Editors) Adv. Drug Delivery Rev.: CancerNanotechnology; Volume 66 ed.; Elsevier: 2014.).

In some embodiments, the microRNA or anti-miRNA sequence is at leastabout 3 nucleotide in length. In some embodiments, the miRNA moleculehas a length of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotidesor more. IN some embodiments, an anit-miRNA or an antagomir of a miRNAmolecule is at least about 6 nucleotides in length. In some embodiments,the antagomir of a miRNA molecule has a length of at least about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 nucleotides or more.

In some embodiments, to interfere oncogenic miRNA to regress brain tumorgrowth, the RNA nanostructure molecule contains anti-miRNA that silencesoncogenic miRNAs, including but not limited to, miR-9, miR-10b, miR-21,miR-17, and miR-26. In some embodiments, to rescue down-regulated tumorsuppressive miRNAs, RNA nanostructure introduces includes tumorsuppressive miRNAs, including but not limited to, let-7a, miR-10b,miR-25, miR-34a, miR-124, miR-145, and miR-181b. MiRNA sequences arelisted below:

miR-9: (SEQ ID NO: 8) 5′-UCUUUGGUUA UCUAGCUGUA UG-3′ miR-10b:(SEQ ID NO: 9) 5′-UACCCUGUAGAACCGAAUUUGUG-3′ miR-26a: (SEQ ID NO: 10)5′-UUCAAGUAAUCCAGGAUAGGCU-3′ let-7a: (SEQ ID NO: 11)5′-UGAGGUAGUAGGUUGUAUAGUU-3′ miR-25: (SEQ ID NO: 12)5′-AGGCGGAGACUUGGGCAAUUG-3′ miR-34a: (SEQ ID NO: 13)5′-UGGCAGUGUCUUAGCUGGUUGU-3′ miR-124: (SEQ ID NO: 14)5′-CGUGUUCACAGCGGACCUUGAU-3′ miR-145: (SEQ ID NO: 15)5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ miR-181b: (SEQ ID NO: 16)5′-AACAUUCAUUGCUGUCGGUGGGU-3′

In some embodiments, the microRNA includes a locked nucleic acid (LNA)sequence. In some embodiments, the microRNA is a LNA-anti-miR21 sequence

(SEQ ID NO: 7) 5′-+G+A+T+A+A+G+C+T CTC CCG GCC GCC ATG GCC GCG GGA T-3′(underlined sequence is 8-mer anti-miR21 LNA, and “+” denotes LNAsequence). In some embodiments, the RNA nanostructure contains a strandLNA17_sph1: 5′-+A+G+C+A+C+T+T+TCTCCCGGCCGCCATGGCCGCGGGAT-3′ (SEQ ID NO:17) (“+” denotes LNA sequence.) In another embodiment, the RNAnanostructure contains a strand of LNA19a_sph1:5′-+A+T+T+T+G+C+A+CCTCCCGGCCGCCATGGCCGCGGGAT-3′ (SEQ ID NO: 18) (“+”denotes LNA sequence.).

In some embodiments, the present disclosure provides inhibitors ofmiRNAs (e.g., anti-miR-21). Compositions comprising such inhibitors andmethods for inhibiting miR-21 using such inhibitors are also disclosedherein. Any miRNA inhibitor may be used alone, or with other miRNAinhibitor(s) known in the art. In some embodiments, the miRNA inhibitorcomprises an antisense molecule. In some embodiments, the antisensemolecule could be a single or a double stranded sequence. Examples ofantisense molecule include, but are not limited to, siRNAs,triple-helix-forming agents, ribozymes, RNAi, synthetic peptide nucleicacids (PNAs), antigenes (agRNAs), LNA/DNA copolymers, small moleculechemical compounds, and antisense oligonucleotides.

Further provided, in some embodiments of the presently disclosed subjectmatter, is a nucleic acid composition. The composition includes atherapeutically effective amount of the artificial RNA nanostructuremolecule as disclosed above. In some embodiments, the compositionincludes a pharmaceutically acceptable carrier.

Still further, the presently disclosed subject matter, in someembodiments, provides a nanoparticle delivery system. The deliverysystem includes the artificial RNA nanostructure molecule as disclosedabove. In some embodiments, the nanoparticle delivery system furtherincludes a pharmaceutically acceptable carrier.

In another aspect, the presently disclosed subject matter provides, insome embodiments, a method of treating a brain tumor in a subject havingor at risk of developing a brain tumor. The method includesadministering to the subject a therapeutically effective amount of acomposition comprising an artificial RNA nanostructure molecule asdisclosed above and herein. In some embodiments, the compositionincludes a pharmaceutically acceptable carrier.

Further, in some embodiments, the present disclosure provides a methodof preventing brain tumor recurrence a subject having or at risk ofhaving brain tumor recurrence. The method includes administering to thesubject a therapeutically effective amount of a composition comprisingan artificial RNA nanostructure molecule as disclosed above and herein.In some embodiments, the composition includes a pharmaceuticallyacceptable carrier.

Brain tumors are a very serious and are among the most difficult totreat, with a very short survival in patients, despite administration ofthe optimal treatment available. The very unique biological environmentof the brain, as separated by the blood-cerebrospinal fluid barrier(BCFB), significantly contributes to a range of site-specific cancers inthis organ that require alternative treatment than those cancers of theremaining human body. Treatment consists primarily of surgical removaland radiation therapy; chemotherapy is also used, but the range ofsuitable chemotherapeutic agents is limited, perhaps because mosttherapeutic agents do not penetrate the blood-brain barrier adequatelyto treat brain tumors. Using known chemotherapeutics along with surgeryand radiation rarely extends survival much beyond that produced bysurgery and radiation alone. Thus improved therapeutic options areneeded for brain tumors.

In some embodiments, the brain tumor is a glioma. Gliomas are a commontype of brain tumor. They arise from the supportive neuronal tissuecomprised of glial cells (hence the name glioma), which maintain theposition and function of neurons. In some embodiments, gliomas areclassified according to the type of glial cells they resemble:astrocytomas (including glioblastomas) resemble star-shaped astrocyteglial cells, oligodendrogliomas resemble oligodendrocyte glial cells;and ependymomas resemble ependymal glial cells that form the lining offluid cavities in the brain. In some embodiments, a tumor may contain amixture of these cell types, and would be referred to as a mixed glioma.

As disclosed herein, in some embodiments, the brain tumor is aglioblastoma. Glioblastomas is the most common primary brain tumors inadults and are also one of the most deadly cancers (1). The mediansurvival of patients with glioblastoma is less than 15 months. The poorprognosis is primarily due to tumor recurrence, which is thought tooriginate from a subset of cancer stem cells that survive the primarytreatments. Recent studies suggested that glioblastoma stem cellsurvived the therapeutic stresses and become more aggressive when theyrecur, developing resistance to the primary chemotherapy. In someembodiment, the presently disclosed subject matter provides a method ofadministering the RNA nanostructure composition as disclosed herein toboth brain tumor cells and glioblastoma stem cells to treat the primarybrain tumor and prevent tumor recurrence.

In some embodiments, the method(s) as disclosed herein includesadministering to the subject a therapeutically effective amount acomposition. The composition includes an artificial RNA nanostructuremolecule, wherein the molecule includes a multiple branched RNA junctionmotif comprising at least one RNA oligonucleotide, a brain tumortargeting module coupled to an RNA junction motif, and at least onetherapeutic agent coupled to the RNA junction motif. In someembodiments, the composition further includes a pharmaceuticallyacceptable carrier. In some embodiments, the bioactive agent comprises atherapeutic agent. In some embodiments, the RNA oligonucleotidecomprises at least one chemical modification at the 2′ position.Non-limiting examples of the modification comprises 2′Fluoro, 2′ Amine,2′O-Methyl, or a combination thereof. In some embodiments, the motif isa three-branched RNA junction motif. An non-limiting example of thethree-branched RNA junction motif comprises a packaging RNA (pRNA)three-way junction (3WJ) motif. In some embodiments, the diameter of themolecule is at least about 40 nm or less. In some embodiments, themolecule has a zeta potential ranging from about −50 mV to about 50 mV.In some embodiments, the multiple branched RNA includes a nucleotide5′-UUG CCA UGU GUA UGU GGG AUC CCG CGG CCA UGG CGG CCG GGA G-3′ (SEQ IDNO: 6). In another embodiment, the multiple branched RNA comprisessequence 5′-GATAAGCT CTC CCG GCC GCC ATG GCC GCG GGA T-3′ (SEQ ID NO:7). In some embodiments, a branch of the three-branched RNA junctionmotif includes at least one of an a3WJ RNA module (SEQ ID NO: 1); a b3WJRNA module (SEQ ID NO: 2); or a c3WJ RNA module (SEQ ID NO: 3).

In some embodiments, the brain tumor targeting module in the method(s)of the presently disclosed subject matter includes a ligand that bindsto at least one brain tumor cell surface marker. In some embodiments,the ligand binds to a folate receptor, an EGFR, a transferrin receptor,an RGD, or a combination thereof. In some embodiments, the ligandcomprises an aptamer. In some embodiments, the targeting modulecomprises a folate. Non-limiting examples of folate include folic acid,5-methyltetrahydro folate, 5-formyltetrahydrofolate, dihydrofolate,tetrahydrofolate, or other folate compounds.

In some embodiments, the therapeutic agents in the method(s) of thepresently disclosed subject matter includes a drug, a fluorescent dye, achemical, or a combination thereof. Further, the therapeutic agentincludes a siRNA, a miRNA, an anti-miRNA, a ribozyme RNA, an antisenseRNA, or a combination thereof. In some embodiments, the therapeuticagent is directed to a brain tumor marker. In some embodiments, thetherapeutic agent is a siRNA sequence, or a microRNA sequence. In someembodiments, the microRNA sequence is at least 6 nucleotide in length.Non-limiting example of the microRNA is a locked nucleic acid (LNA)sequence. An example of the LNA sequence is a LNA-miR21 sequence asdescribed herein. In some embodiments, the siRNA binds to a mRNAsequence of a gene that promotes tumorigenesis, angiogenesis, cellproliferation, or a combination thereof, in the brain or spinal cord. Insome embodiments, the siRNA binds to a mRNA molecule that encodes aprotein including pro-tumorigenic pathway proteins, pro-angiogenesispathway proteins, pro-cell proliferation pathway proteins,anti-apoptotic pathway proteins, or a combination thereof. In someembodiments, the mRNA molecule encodes a protein including VEGF pathwayproteins, EGFR pathway proteins, MGMT pathway proteins, Rafl pathwayproteins, MMP pathway proteins, mTOR pathway proteins, TGFβ pathwayproteins, or Cox-2 pathway proteins, or a combination thereof. In someembodiments, the protein includes VEGF, EGFR, POK, AKT, AGT, RAF, RAS,MAPK, ERK, MGMT, MMP-2, MMP-9, PDGF, PDGFR, IGF-I, HGF, mTOR, Cox-2 orTGFβ1.

Further, in some embodiments of the methods, the present subject matterrelates to a method to target and deliver therapeutic siRNA to braintumors using FA-conjugated pRNA-3WJ RNP. First, intracranial tumorxenograft models in mice was established and then systemicallyadministered RNPs through the tail vein. Based on fluorescence imaging,It is demonstrated that the pRNA-3WJ RNP efficiently targeted andinternalized into brain tumor cells through FR-mediated endocytosis withlittle or no accumulation in adjacent healthy brain cells. Genesilencing by the RNPs was also demonstrated within the luciferase geneexpressing brain tumors. More importantly, pRNA-3WJ RNPs were alsocapable of targeting brain tumor stem cells derived from a humanpatient. The data demonstrate that artificially engineered RNPs canspecifically target brain tumor cells, including glioblastoma stemcells, and deliver functional siRNA and therapeutic microRNAs (miRNAs)(21).

The term “treatment” and “prophylaxis” as used herein is to beconsidered in its broadest context. The term “treatment” does notnecessarily imply that a host is treated until total recovery.Similarly, “prophylaxis” does not necessarily mean that the subject willnot eventually contract a disease condition. Accordingly, treatment andprophylaxis include amelioration of the symptoms of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition. The term “prophylaxis” may be considered asreducing the severity of onset of a particular condition. “Treatment”may also reduce the severity of an existing condition.

The term “pharmaceutically acceptable carrier” as used herein refers toa diluent, adjuvant, excipient, or vehicle with which a heterodimericprobe of the disclosure is administered and which is approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. Such pharmaceutical carrierscan be liquids, such as water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, and the like. The pharmaceutical carriers canbe saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea, and the like. When administered to a patient, theheterodimeric probe and pharmaceutically acceptable carriers can besterile. Water is a useful carrier when the heterodimeric probe isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical carriers also includeexcipients such as glucose, lactose, sucrose, glycerol monostearate,sodium chloride, glycerol, propylene, glycol, water, ethanol, and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thepresent compositions advantageously may take the form of solutions,emulsion, sustained release formulations, or any other form suitable foruse.

The term “therapeutically effective amount,” as used herein, refers tothe amount of a composition containing administered to a patient alreadysuffering from a disease, condition, or disorder, sufficient to cure orat least partially arrest, or relieve to some extent one or more of thesymptoms of the disease, disorder, or condition being treated. Theeffectiveness of such compositions depend upon conditions including, butnot limited to, the severity and course of the disease, disorder, orcondition, previous therapy, the patient's health status and response tothe drugs, and the judgment of the treating physician. By way of exampleonly, therapeutically effective amounts may be determined by routineexperimentation, including but not limited to a dose escalation clinicaltrial.

The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration; the route of administration; therate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of acompound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. If desired, the effective daily dose can bedivided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, the term “subject” refers to a target of administrationof the pharmaceutical composition. The subject of the herein disclosedmethods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. Thus, the subject of the herein disclosedmethods can be a human or non-human. Thus, veterinary therapeutic usesare provided in accordance with the presently disclosed subject matter.As such, the presently disclosed subject matter provides foradministration to mammals such as humans and non-human primates, as wellas those mammals of importance due to being endangered, such as Siberiantigers; of economic importance, such as animals raised on farms forconsumption by humans; and/or animals of social importance to humans,such as animals kept as pets or in zoos. Examples of such animalsinclude but are not limited to: carnivores such as cats and dogs; swine,including pigs, hogs, and wild boars; ruminants and/or ungulates such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels; rabbits,guinea pigs, and rodents. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like. The term does not denote aparticular age or sex.

Suitable methods for administering to a subject an effective amount ofthe composition in accordance with the methods of the present inventioninclude but are not limited to systemic administration, parenteraladministration (including intravascular, intramuscular, intraarterialadministration), oral delivery, buccal delivery, subcutaneousadministration, inhalation, intratracheal installation, surgicalimplantation, transdermal delivery, local injection, and hyper-velocityinjection/bombardment. Where applicable, continuous infusion can enhancedrug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082).

The particular mode of drug administration used in accordance with themethods of the present invention depends on various factors, includingbut not limited to the vector and/or drug carrier employed, the severityof the condition to be treated, and mechanisms for metabolism or removalof the drug following administration.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the presently disclosed subject matter.

EXAMPLES Example 1

Glioblastoma is one of the most deadly cancers. Systemic siRNAadministration to treat glioblastoma patients requires a robust andefficient delivery platform without immunogenicity. This example reportthe application of RNA nanotechnology based on pRNA 3-way-junction (3WJ)of bacteriophage phi29 for glioblastoma targeting. Multivalent folate(FA)-conjugated RNA nanoparticles were constructed to harbor siRNA. Theresulted FA-pRNA-3WJ RNA nanoparticle (RNP) specifically targeted andentered human malignant glioblastoma cells in vitro and intracranialglioblastoma xenografts in vivo. Systemically injected FA-pRNA-3WJ RNPssuccessfully targeted and delivered siRNA into brain tumor cells inmice, and efficiently reduced luciferase reporter gene expression(4-fold lower than control). The FA-pRNA-3WJ RNA nanoparticles were alsodemonstrated to target both human patient-derived glioblastoma stemcells, which are thought to be responsible for tumor initiation, drugresistance and deadly recurrence, and the derived brain tumor in mousemodel without accumulation in adjacent normal brain cells, nor othermajor internal organs. The results of this study may promise asuccessful clinical application of pRNA-3WJ RNP for specific delivery oftherapeutics such as siRNA, microRNA and/or chemotherapeutic drugs intoglioblastoma cells without inflicting collateral damage to healthytissues.

Results and Discussion

This study was to assess application of pRNA-3WJ RNP for systemicdelivery of therapeutic RNA, such as siRNA and miRNA, into brain tumorsin a mouse model system. For targeted delivery of siRNA into braintumors, a multifunctional RNP was constructed, as previously described(7, 12, 13, 22), using a scaffold based on pRNA sequences of phi29bacteriophage with slight modifications (see Materials and Methods).Three RNA modules individually transcribed in vitro or synthesizedchemically were mixed at equal molar ratio and formed three-branched RNPvia one-step self-assembly. Each RNA module was designed to carry afunctional moiety: 1) FA as the FR targeting ligand; 2) fluorophoreAlexa647 as the imaging agent; and 3) luciferase siRNA as the genesilencing functional moiety or scrambled RNA as a negative control (FIG.1A) (7, 12, 13, 22). The resulting RNP was named FA-pRNA-3WJ-si(luc)RNP. Observation of the self-assembled FA-pRNA-3WJ-si(luc) RNP underatomic force microscopy (AFM) revealed the formation of homogeneousthree-branched architectures with 3WJ core in the center (FIG. 1B),confirming the previous reports that modifications on each RNA moduledid not abrogate the shape-controlled self-assembly to retain thepRNA-3WJ core structure essential for homogeneous uniformed RNPformation. Dynamic light scattering (DLS) determined averagehydrodynamic diameters of FA-pRNA-3WJ-si(luc) RNP to be 5.2±1.2 nm (FIG.1C), which was smaller than the predicted size (10×4×2 nm) calculated byRNA folding software based on expected duplex helix parameters and basepair lengths of the three individual RNA modules. The discrepancybetween DLS measurement and computational prediction implies that eachprotruded branch of FA-pRNA-3WJ-si(luc) RNP was avoided from averagingthree dimensions due to rapid motion of nanoparticles in solution.Another factor that needs to be addressed for successful systemic invivo application of nanoparticles is freedom from aggregation to avoidrapid clearance from the body and diminished specific interactionbetween the conjugated ligand and cellular target receptors. Aggregationdepends largely on the surface charge of nanoparticles and the surfaceof RNA is indeed highly charged. Aggregation will also change thesurface charge proportional to the extent of size increase. To determinethe aggregation extent, FA-pRNA-3WJ-si(luc) RNP was subjected to zetapotential analysis to measure the particle surface charge. Zetapotential of FA-pRNA-3WJ-si(luc) RNP in PBS solution was measured as asingle peak at −15.8±5.6 mV (FIG. 1D), indicating that mostFA-pRNA-3WJ-si(luc) RNP exist as a single form without aggregation.These physical properties favor the FA-pRNA-3WJ-si(luc) RNP for systemicin vivo application.

Human glioblastoma cells are known to overexpress FR, while normal braincells show no FR expression (17-19). To determine the specificrecognition and binding capability of FA-pRNA-3WJ-si(luc) RNP towardshuman glioblastoma cells, firstly association of FA-Alexa647-pRNA-3WJwith U87EGFRvIII cell was tested in vitro in comparison to FA-freecontrol RNP (Alexa647-pRNA-3WJ). Flow cytometry analysis showed a higherassociation of FA-Alexa647-pRNA-3WJ with U87EGFRvIII cells (63.1±4.5%)than that of Alexa647-pRNA-3WJ (40.3±3.7%) (student t-test, p<0.001,n=4) (FIG. 2A). When FRs of U87EGFRvIII cells were pre-masked byincubating with 1 mM free-folate for 1 hr of culture before the RNPbinding, the association between FA-Alexa647-pRNA-3WJ and U87EGFRvIIIcells was decreased to an extent similar to the negative controlAlexa647-pRNA-3WJ (FIG. 5), indicating that the association betweenFA-Alexa647-pRNA-3WJ and U87EGFRvIII cells was FR dependent. TheFR-mediated specific binding of FA-Alexa647-pRNA-3WJ to U87EGFRvIIIcells was further confirmed by visualizing the Alexa647 signal fromsurface-cultured U87EGFRvIII cells treated with FA-Alexa647-pRNA-3WJ RNPunder confocal fluorescence microscope. Higher fluorescence intensity ofAlexa647 dye was observed from U87EGFRvIII cells treated withFA-Alexa647-pRNA-3WJ than those with control RNP (Alexa647-pRNA-3WJ)(FIG. 2B). Again, the FA-dependent association of FA-Alexa647-pRNA-3WJRNP was abolished by pre-treatment of U87EGFRvIII cells with 1 mM freefolate in culture medium (FIG. 2B). The FR-mediated specific associationbetween FA-Alexa647-pRNA-3WJ RNP and human glioblastoma cell was alsoobserved with other glioblastoma cell lines including T98G (FIG. 6).Taken together, FA-conjugated pRNA-3WJ RNP has the capability torecognize and bind to human brain tumor cells through FR.

Next, we tested whether FA-pRNA-3WJ RNP can specifically target tumorcells in vivo using an orthotropic mouse model of glioblastoma. On the14th day post U87EGFRvIII cell implantation into nude mouse brain,intracranial tumor growth in mice was determined by MRI (FIG. 2C, top)and randomly separated into three groups for injection of PBS,Alexa647-pRNA-3WJ as two negative controls and FA-Alexa647-pRNA-3WJ asexperimental. Each group of mice (n=4) was injected via tail vein (1mg/kg of RNP in 100 μL of PBS). Fifteen hours post injection, the micebrains were dissected and subjected to fluorescence imaging to detectthe Alexa647 signal from RNP. A higher fluorescence signal of Alexa647was observed in the brains of mice injected with FA-Alexa647-pRNA-3WJthan that in the mice brains injected with control RNP(Alexa647-pRNA-3WJ) (FIG. 2C). ANOVA analysis on the fluorescenceintensity from each group (n=4) normalized by their tumor volumes(Alexa647 intensity/tumor volume) confirmed the significant increase inaverage fluorescence intensity in the mouse brains treated withFA-Alexa647-pRNA-3WJ (2.052±0.416, s.e.m.) compared to Alexa647-pRNA-3WJ(1.014±0.279, s.e.m.) (p=0.019) with respect to PBS (1.000±0.298,s.e.m.) (FIG. 2D). The brain tumor region was frozen sectioned (10 μmthick) and further examined under a fluorescence confocal microscope. Itrevealed that FA-Alexa647-pRNA-3WJ RNP was mostly associated withcounterstained brain tumor cells (Supplementary FIG. 7). These in vivodata strongly indicated that systemically injected FA-Alexa647-pRNA-3WJRNP can travel to brain tissue, and successfully recognize and bindhuman glioblastoma cells through FA-FR interaction, rather than randomlydistribute throughout the entire brain tissues.

After binding to target glioblastoma cells, RNP needs to internalize todeliver its cargo, siRNA, for successful target gene silencing, which isthe most critical property for any nanoparticle to claim its therapeuticapplication. In order to test whether siRNA-loaded FA-3WJ RNP cansilence the target gene in glioblastoma cells in mouse brain aftersystemic administration, we set up a luciferase-based gene expressionreporter system by implanting luciferase gene-expressing U87EGFRvIIIcells (U87EGFRvIII-Luc) in mouse brain. For a preliminary in vitro test,U87EGFRvIII-Luc cells were incubated for 72 hrs in culture mediumcontaining a range between 0 and 400 nM of FA-pRNA-3WJ-si(Luc) orscrambled RNA-conjugated control FA-pRNA-3WJ-si(Scrm) RNPs without anytransfection agent. After 72 hrs, FA-pRNA-3WJ-si(Luc) clearly reducedluciferase activity in a concentration dependent manner. At 400 nM,average luciferase activity in U87EGFRvIII-Luc cells incubated withFA-pRNA-3WJ-si(Luc) was decreased about five folds (0.214±0.210, s.e.m.)with respect to 0 nM. However, FA-pRNA-3WJ-si(Scrm) did notsignificantly reduce luciferase activity in the cells (0.876±0.056,s.e.m.) compared to 0 nM. The difference of luciferase activity at 400nM between FA-pRNA-3WJ-si(Luc) and FA-pRNA-3WJ-si(Scrm) wasstatistically significant (p=0.006) (FIG. 3A). For in vivo test,intracranial tumor in mice was induced by implanting U87EGFRvIII-Luccells. Bioluminescence signal measured from the resulted brain tumor isexpected to correlate with tumor growth. When a group of braintumor-bearing mice (n=5) were systemically injected withFA-pRNA-3WJ-si(Scrm) (1 mg/kg in 100 μL of PBS) for a total of threetimes over 6 days, the luciferase activity rapidly increased as thetumor grew indicating no effect of the control RNP on luciferase geneexpression. However, luciferase activity from the group of mice (n=5)injected with FA-pRNA-3WJ-si(Luc) was observed to increase very slowlyover time (FIG. 3B). After 3 injections, the luciferase activity fromthe mice injected with FA-pRNA-3WJ-si(Luc) was significantly lower(p=0.007) than that from the control group mice injected withFA-pRNA-3WJ-si(Scrm). The luciferase activity from the tested mice atday 13 post tumor implantation was mostly lower than that from the micetreated with FA-pRNA-3WJ-si(Scrm) (FIG. 3C). However, Mill revealed thatthe relative tumor size between those two groups was statisticallyinsignificant (1.160±0.352 mm³, s.e.m. with respect to 1.000±0.300 mm³in negative control group) (p=0.468, n=5) (FIG. 3D). When theirluciferase activity was normalized by the tumor volumes, the relativelyaveraged luciferase activity over tumor volumes from the mice treatedwith FA-pRNA-3WJ-si(Luc) (0.255±0.040 Luminescence Radiance[p/s/cm²/sr]/tumor volume [mm³], s.e.m.) was significantly lowercompared to the control mice group treated with FA-pRNA-3WJ-si(Scrm)(1.000±0.410 Luminescence Radiance [p/s/cm²/sr]/tumor volume [mm³],s.e.m.) (p=0.015, n=5) (FIG. 4E). These data strongly indicated thatFA-pRNA-3WJ RNP not only specifically targeted glioblastoma cells, butalso successfully internalized into the cells and delivered the cargosiRNA. The delivered siRNA, more importantly, remained functionallyintact for the whole time of systemic delivery, confirming bothstability and therapeutic efficacy of the FA-pRNA-3WJ RNPs. The datasuccessfully demonstrated the therapeutic usability as a siRNA deliverysystem for glioblastoma treatment.

In clinical settings, glioblastomas are notorious for their frequentrecurrence with increased aggressiveness after initial therapy,resulting in poor survival rates. It has been hypothesized thatglioblastoma stem cells tend to survive the initial treatment and inducetumor recurrence, meaning that any therapeutic strategy lacking theability to kill glioblastoma stem cells would not prevent recurrences(23). The potential of FA-pRNA-3WJ RNPs to target glioblastoma stemcells and their derived tumor cells was investigated. We used humanglioblastoma patient-derived primary neurosphere cells, named “1123”,which has been shown to possess stem cell-like characteristics includinga high level of CD44 expression, self-renewal capability andtumorigenicity when implanted in mouse brain (24-26). First, the CD44⁺1123 cells, maintained in serum-free sphere culture medium, wereincubated in vitro with 200 nM of either FA-Alexa647-pRNA-3WJ orAlexa647-pRNA-3WJ RNPs. Flow cytometry analysis revealed higherFA-Alexa647-pRNA-3WJ binding to the 1123 cells than control RNP(Alexa647-pRNA-3WJ) (FIG. 4A). Compared to PBS-treated 1123 cells,33.2±0.8% of CD44⁺ 1123 cells were positively associated withFA-Alexa647-pRNA-3WJ RNP. However, Alexa647-pRNA-3WJ control RNP wasassociated with only 12.7±0.4% of CD44⁺ 1123 cells. The differencebetween FA-Alexa647-pRNA-3WJ and Alexa647-pRNA-3WJ was statisticallysignificant (p<0.0001).

For systemic assessment, a group of mice was then implanted with 1123cells to induce intracranial brain tumor. Determined by MRI, micebearing a similar size of brain tumors were then injected with PBS,Alexa647-pRNA-3WJ or FA-Alexa647-pRNA-3WJ RNPs in 100 μL of PBS throughthe tail vein. Fifteen hours post injection, the brains were dissectedout and subjected to fluorescence imaging. Higher accumulation ofFA-Alexa647-pRNA-3WJ RNP was observed in the tumor region, while lessaccumulation of Alexa647 signal was observed from the brains treatedwith either control RNP (Alexa647-pRNA-3WJ) or PBS (FIG. 4B). When twodifferent dosages of FA-Alexa647-pRNA-3WJ RNPs (20 or 100 μg/mouse) weretested in a group of mice bearing small sized tumors, fluorescencesignals were proportional to the amount of RNPs injected (SupplementaryFIG. 8). These observations suggests that FA-Alexa647-pRNA-3WJ RNP canalso recognize and target human glioblastoma stem cells and theirderived tumor cells through FA-FR specific interaction. Throughout thesestudies, a fluorescence signal from the groups treated with FA-free3WJ-pRNA control RNP was also observed although the extents were alwayslower than the groups treated with FA-3WJ-pRNA RNP. This might beexplained by the nature of tumor induced from human patient derived stemcell-like glioblastoma cells, in which the aggressive hypervasculatureleaves a large portion of blood vessels as “leaky” as they are poorlyfinished before forming a tight junction of the BBB, also called the EPR(enhanced permeability and retention) effect (27).

To assess the biodistribution profile of the pRNA-3WJ RNP throughout thebody after systemic administration, major internal organs, includingheart, lung, liver, spleen and kidney, were also collected together withbrain and subjected to fluorescence imaging. Compared to brain, nosignificant fluorescence signal was detected from the internal organsexcept kidney (FIG. 4C). The biodistribution profile of FA-pRNA-3WJ RNPafter its systemic administration was consistent with the previousreport (28), in that FA-conjugated drugs that failed to target tumorcells are rapidly cleared from a mouse body (t_(1/2)<10 min) through thekidney, reducing the safety concern of unbound pRNA-3WJ nanoparticlescirculating in the blood (7,12-14).

For successful clinical application of pRNA-3WJ RNP for humanglioblastoma detection and treatment, it was critical to evaluate itscapability to access brain tumor cells by discriminating them fromadjacent normal brain cells, and to have favorable biodistribution. Toaddress those two goals, the most critical checkpoints deciding thedrugability of the pRNA-3WJ nanoparticles, we employed an orthotropicintracranial glioblastoma model system in mice. According to ourobservations, it was clear that FA-conjugated FA-pRNA-3WJ RNP can targethuman glioblastoma cells through FA-FR specific interaction-mediatedendocytosis by distinguishing glioma cells from adjacent normal braincells. A series of in vitro experiments indicated that such targeting invivo was not obviously a result of non-specific accumulation for tworeasons: 1) association of FA-pRNA-3WJ RNP with glioblastoma cells wasligand-dependent; and 2) the association was mediated through FA-FRspecific interaction, since free folate pre-treatment interfered withthe specific interaction between FA and FRs on the targeted cells. Thissuggests that FA-pRNA-3WJ RNP can target and accumulate in FR⁺glioblastoma cells. Taken together with the fact that our brain imagingdata were collected 15 hrs post injection of FA-3WJ RNA nanoparticlesand the luciferase gene silencing effect was seen for days, these datasuggest that FA-pRNA-3WJ RNP can survive in the body by retaining thechemical integrity of cargo siRNA until it reaches the brain. Mostimportantly, the therapeutic delivery by the FA-3WJ RNA nanoparticleswas clearly demonstrated by targeting endogenous luciferase mRNAs as areporter system (FIG. 3). The decreased luciferase activity observedfrom a group of mice injected with FA-3WJ-pRNA-si(Luc) RNP clearlyanswered questions towards the capability of the FA-3WJ RNPregarding: 1) specific targeting to brain tumor cells; 2)internalization into brain tumor cells; and 3) releasing the functionalmoiety (siRNA against luciferase mRNA). In addition, the targetingcapability of pRNA-3WJ nanoparticles for both brain tumor cells andglioblastoma stem cells through a FA-FR mediated manner will overcomethe weak point of conventional brain tumor therapies which largelyrelies on surgical debulking and less-specific toxic drugs withradiation. In summary, our current study successfully demonstrated thedrugability of FA-conjugated pRNA-3WJ RNP as therapeutic gene deliveryfor clinical applications to meet the urgent need of new strategies totarget and kill both glioblastoma stem cells and their derived tumorcells. Due to the ease and flexibility of modification of each RNAmodule, any drug conjugation and siRNA can be loaded to the RNP astherapeutic functionalities. Recently, microRNAs have been found toinvolve in pathological process in glioblastoma making them as promisingtherapeutic targets (29-32). Since size and working mechanism of miRNAsare similar to those of siRNAs (21), therapeutic miRNAs also can beconsidered to be loaded onto the pRNA-3WJ-RNP.

Methods

Construction of FA-Alexa647-pRNA-3WJ-si(Luc) RNP

Multifunctional pRNA-3WJ RNP was prepared as previously described (7,12, 13, 22) with slight modifications. In brief, three RNA modules,named a_(3WJ)(5′-UUGCCAUGUGUAUGUGGG-3′ (SEQ ID NO: 1)),b_(3WJ)(5′-CCCACAUACUUUGUUGAUCC-3′ (SEQ ID NO: 2)), andc_(3WJ)(5′-GGAUCAAUCAUGGCAA-3′ (SEQ ID NO: 3)), were transcribed invitro or synthesized chemically using 2′-F modified nucleotides andpurified separately to homogeneity. For the current study, each RNAmodule was further modified as following: module a_(3WJ) was extendedwith luciferase siRNA sequences sense: 5′-CUUACGCUGAGUACUUCGAUU-3′ (SEQID NO: 4) and anti-sense: 5′-UCGAAGUACUCAGCGUAAGUU-3′ (SEQ ID NO: 5), orscrambled as negative control; module b_(3WJ) was conjugated with FA atthe 3′ end; and module c_(3WJ) was conjugated with fluorophore Alexa647(Alexa Fluor® 647, Invitrogen) at the 3′ end. The three RNA modules weremixed at equal molar ratio to form one-step self-assembly. Theself-assembled FA-Alexa647-pRNA-3WJ-si(Luc) RNPs were purified from 6 Murea-containing PAGE and frozen at −80° C. after reconstituted in PBS.To obtain the designated concentration for each experiment, the RNPswere further diluted in PBS before use.

Characterization of Self-Assembled FA-Alexa647-pRNA-3WJ-si(Luc) RNP

Three dimensional structure and shape of the final form ofself-assembled FA-Alexa647-pRNA-3WJ-si(Luc) RNP was analyzed by atomicforce microscopy (AFM) imaging as described previously.^(7,12,13,22)Apparent hydrodynamic sizes and zeta potential of pre-assembledFA-Alexa647-pRNA-3WJ-si(Luc) RNP (1.5 μM) in PBS buffer was measured byZetasizer nano-ZS (Malvern Instrument) at 25° C. The laser wavelengthwas 633 nm. The data were obtained from three independent measurements.

Human Glioblastoma Cells and Human Patient-Derived Glioblastoma StemCells

Human glioblastoma cells, U87EGFRvIII and U87EGFRvIII-Luc (expressingluciferase reporter gene), were obtained from Dr. Webster Cavenee(Ludwig Cancer Institute, San Diego, Calif.). Both cells were maintainedin DMEM/10% FBS/penicillin/streptomycin. Human glioblastomapatient-derived glioblastoma stem cell “1123” was cultured in DMEM/F12(Invitrogen) supplemented with B27 (1:50), heparin (5 mg/mL), basic FGF(bFGF) (20 ng/mL), and EGF (20 ng/mL). Growth factors (bFGF and EGF)were added twice a week (24).

Intracranial Human Glioblastoma Xenografts from Human Glioblastoma Cellsand Human Patient-Derived Glioblastoma Stem Cells

Six weeks old athymic female nu/nu mice (Jackson Laboratory, Bar Harbor,Me.) were housed and handled in accordance with the Subcommittee onResearch Animal Care of the Ohio State University guidelines approved bythe Institutional Review Board. All mice were fed folate-free diet(Harlan, Indianapolis, Ind.) for at least two weeks before tumorimplantation. Intracranial human glioblastoma xenograft tumor wasinduced by implanting human glioblastoma cell U87EGFRvIII or humanpatient-derived glioblastoma stem cells (1×10⁵ cells per mouse), asdescribed previously (33). Two weeks post intracranial tumorimplantation, the location and size of the implanted tumors weredetermined by magnetic resonance imaging (MM).

Magnetic Resonance Imaging (MRI) for Location and Size of ImplantedBrain Tumor in Mice

On the indicated day post-surgery of intracranial tumor injection, thelocation and size of the implanted tumors were determined by magneticresonance imaging (MRI). Mouse was anesthetized with 2.5% isofluranemixed with 1 L/min carbogen (95% O₂ with 5% CO₂), then maintained with1% isoflurane thereafter. Maintaining core temperature using a warmwater bath, imaging was performed using a Bruker Biospin 94/30 magnet(Bruker Biospin, Karlsruhe, Germany). Mice were injected with Magnevist,gadolinium-based contrast agent (Bayer Health Care Pharmaceuticals,Wayne, N.J.) by an i.p. administration with 0.5 mmol/kg dose, thenpositioned in the magnet. T2-weighted RARE imaging was collected using asequence (TR=3524 ms, TE=36 ms, rare factor=8, navgs=2, FOV=20×20 mm,0.5 mm slice thickness). Region-of-interest (ROI) was manually outlinedbased on contrast in signal intensity between brain and tumor tissue.

Fluorescence Confocal Microscopy for In Vitro and In Vivo RNP Binding

For the in vitro targeting test of pRNA-3WJ RNP, 2×10³ of U87EGFRvIII(malignant human glioblastoma) cells in 200 μL were plated in Lab-TekII8-well chamber slide (Nunc, Rochester, N.Y.). The next day, the cellswere washed with PBS and incubated with 200 nM of eitherFA-Alexa647-pRNA-3WJ RNP or control RNP (Alexa647-pRNA-3WJ) in 200 μL ofculture media for 2 hrs at 37° C. in a CO₂ incubator. To block cellularFRs by free folate, PBS-washed cells were pre-treated with 1 mM freefolate in 200 μL of culture media for 1 hr at 37° C. in a CO₂ incubatorbefore RNP treatment. After washing with PBS, the RNP-treated cells werefixed in 4% paraformaldehyde (PFA) solution for 2 hrs at 4° C. Thecytoskeleton of the fixed cells was stained by Alexa Fluor 488Phalloidin (Invitrogen, Grand Island, N.Y.) for 30 min at roomtemperature and the nucleus stained with 0.01% DAPI solution for 10 minat room temperature. The cells were then rinsed with PBS for 3×10 minand mounted with PermaFluor Aqueous Mounting Medium (Thermo Scientific).Fluorescence microscopy was performed using Olympus 4-filter-basedFluoView FV1000-Filter Confocal Microscope System (Olympus Corp.) at thewavelengths of 461 nm (for the cell nucleus stained by DAPI), 530 nm(for the cytoskeleton stained by Alexa Fluor 488 Phalloidin) and 665 nm(for the Alexa647). Images were analyzed by Olympus FluoView Viewersoftware ver. 4.0 (Olympus). For in vivo targeting, the brain tumorxenograft collected 15 hrs after RNP injection was fixed in 4% PFA with10% sucrose in PBS overnight at 4° C. and embedded in Tissue-Tek® O.C.T.compound (Sakura Finetek USA, Inc., Torrance, Calif.) for frozensectioning (10 μm thick). The sectioned samples were mounted by ProLong®Gold Antifade Reagent with DAPI (Life Technologies Corporation,Carlsbad, Calif.) overnight. The fluorescent images were obtained usingFluoView FV1000-Filter Confocal Microscope System (Olympus Corp.).

Flow Cytometry for In Vitro RNP Binding

Flow cytometry analysis was performed for in vitro targeting by pRNA-3WJRNP in malignant human glioblastoma (U87EGFRvIII) and glioblastoma stemcells (1123). The cells were plated in 6-well plate one day before RNPbinding. After washing with PBS, the cells were incubated with 200 nM ofeither FA-Alexa647-pRNA-3WJ RNP or Alexa647-pRNA-3WJ RNP in 200 μL ofculture media for 2 hrs at 37° C. in a CO₂ incubator. For blockingcellular FRs by free folate, the PBS-washed cells were pre-treated with1 mM of free folate in 200 μL of culture media for 1 hr in 37° C. CO₂incubator before RNP treatment. After washing with PBS, the cells wereharvested by trypsinization and fixed in 4% PFA solution for 2 hrs at 4°C. The cells were washed with PBS for 3 times at room temperature, thensubjected to Flow Cytometry analysis using BD FACS Aria-III Cell Sorter.The data were analyzed by FlowJo 7.6.1 software.

Systemic Injection of RNPs to Intracranial Human Glioblastoma XenograftTumor Bearing Mice

Based on the MRI evaluation taken one day before RNP injection, a groupof mice bearing similarly sized tumors at similar location was selectedfor systemic injection of RNPs. Designated amount of RNPs (1 mg/kg ofmouse body weight) prepared in 100 μL of PBS were injected through mousetail vein. After 15 hrs of RNP injection, the brains were dissected outand subjected to fluorescence imaging. Tumor volume calculated from MRIwas also used to normalize fluorescence intensity or luciferase activityfor each mouse as described below.

Fluorescence Imaging on Human Glioblastoma Xenograft Mouse Brain Tumor

To investigate the delivery of pRNA-3WJ RNPs in vivo, a brainfluorescence imaging study was performed after tail vein injection intomice bearing brain tumor. The mice were sacrificed by cervicaldislocation under anesthesia 15 hrs post injection, and brains weredissected out of mice. Fluorescence signals were detected from thedissected brains using IVIS Lumina Series III Pre-clinical In VivoImaging System (Perkin Elmer, Waltham, Mass.) with excitation at 640 nmand emission at 660 nm for 2 min exposure. The fluorescence intensitywas expressed as Mean Radiant Efficiency [p/s/cm²/sr]/[μW/cm²], thennormalized by tumor volume (mm³). PBS injected mice were used asfluorescence negative control. Major internal organs together with brainfrom the harvested mice were collected and subjected to fluorescenceimaging for assessment of biodistribution profile study.

Bioluminescence Whole Body Imaging for Luciferase Activity

To investigate the siRNA delivery and silencing effect of pRNA-3WJ RNPsin vivo, U87EGFRvIII-Luc cell-induced brain tumor was prepared into twogroups of mice (n=5). At 5, 7 and 9 days post-surgery, 1 mg/kg of mousebody weight of FA-Alexa647-pRNA-3WJ-si(Luc) RNP (or siScrm as negativecontrol) was injected through the mouse tail vein in 100 μL of PBS.After each injection, mice were subjected to bioluminescence whole bodyimaging to detect the endogenous luciferase expression level. Mice wereinjected with 75 mg/kg Luciferin (Perkin Elmer, Waltham, Mass.), andanesthetized. Bioluminescence from the anesthetized mice was detected byZFOV-24 zoom lens-installed IVIS Lumina Series III Pre-clinical In VivoImaging System (Perkin Elmer, Waltham, Mass.). The luminescenceintensity was expressed as Averaged Radiance [p/s/cm²/sr], thennormalized by tumor volume (mm³).

Statistical Analysis

All statistical analyses comparing groups of mice treated with test andcontrol RNPs were performed by either ANOVA or student t-test. p<0.05was considered significant.

Example 2

This study is to determine the specific recognition and bindingcapability of FA-3WJ-LNA-anti-miR21 RNA nanoparticles (RNP) towardshuman glioblastoma cells (FIG. 9A), firstly FA-specific associationbetween FA-3WJ-LNA-miR21 conjugated with Alexa647 fluorescent dye andhuman patient-derived glioblastoma cell GBM30 was assessed in vitro(FIG. 9B). The nanoparticle contains a stand of

(SEQ ID NO: 7) 5′-+G+A+T+A+A+G+C+T CTC CCG GCC GCC ATG GCC GCG GGA T-3′(underlined sequence is 8-mer anti-miR21 LNA).

The cells plated in 6-well plate one day before RNP binding were washedwith PBS and incubated with 200 nM of FA-3WJ-LNA-miR21-Alexa647 RNP for2 hrs at 37° C. in a CO2 incubator. After three times of washing withPBS, the cells were harvested by trypsinization and fixed in 4% PFAsolution for 2 hrs at 4° C. and subjected to Flow Cytometry analysisusing BD FACS Aria-III Cell Sorter. The cells were identified bystaining actin filaments with Phalloidin-Alexa488. Comparison to FA-freecontrol RNP (3WJ-Alexa647), flow cytometry analysis showed a higherassociation in FA-3WJ-LNA-miR21-Alexa647 (23.1%) (student t-test,p<0.001, n=3). Extra moiety of LNA-miR21 did not affect the specificbinding to the GBM30 cells, since FA-3WJ-Alexa647 RNP showed similarlevel of association (21.4%) to the FA-3WJ-LNA-miR21-Alexa647 (23.1%).

Example 3

In this study, the folate receptor (FR)-dependent specific binding ofFA-3WJ-LNA-miR21-Alexa647 RNP to GBM30 cells was further confirmed byvisualizing the Alexa647 signal from surface-cultured GBM30 cellstreated with FA-3WJ-LNA-miR21-Alexa647 RNP under confocal fluorescencemicroscope (FIG. 10). For the in vitro targeting test ofFA-3WJ-LNA-miR21-Alexa647 RNP, 2×103 of GBM30 cells in 200 μL wereplated in Lab-Tek II 8-well chamber slide. The next day, the cells werewashed with PBS and incubated with 200 nM of eitherFA-3WJ-LNA-miR21-Alexa647 RNP or control RNP (3WJ-Alexa647) in 200 μL ofculture media for 2 hrs at 37° C. in a CO2 incubator. The cytoskeletonof the fixed cells was stained by Alexa Fluor 488 Phalloidin(Invitrogen, Grand Island, N.Y.) for 30 min at room temperature and thenucleus stained with 0.01% DAPI solution for 10 min at room temperature.The cells were then rinsed with PBS for 3×10 min and mounted withPermaFluor Aqueous Mounting Medium (Thermo Scientific). Fluorescencemicroscopy was performed using Olympus 4-filter-based FluoViewFV1000-Filter Confocal Microscope System (Olympus Corp.). Higherfluorescence intensity of Alexa647 dye was observed from GBM30 cellstreated with FA-3WJ-LNA-miR21-Alexa647 RNP than those with control RNP(3WJ-Alexa647) lacking FA. Again, the FA-dependent association was notaffected by the presence of LNA-miR21 sequences, since theFA-3WJ-LNA-miR21-Alexa647 RNP showed comparable association with3WJ-Alexa647. When FRs of GBM30 cells were pre-masked by incubating with1 mM free-folate for 1 hr of culture before the RNP treatment, theassociation between FA-3WJ-LNA-miR21-Alexa647 RNP and GBM30 cells wasabolished to an extent similar to the negative control 3WJ-Alexa647 RNP.Taken together with data shown in FIG. 9, it indicated that theassociation between FA-3WJ-LNA-miR21-Alexa647 RNP and GBM30 cells was FRdependent medicated by the FA conjugated to the RNP. Yellow arrowindicates the specific localization of FA-3WJ-LNA-miR21-Alexa647 RNP inGBM30 cells, which is presented with magnified view in FIG. 11.

Example 4

This study shows the distribution of FA-3WJ-LNA-miR21-Alexa647 RNP inGBM30 cells after 2 hrs of incubation was visualized by confocalfluorescent microscopy (FIG. 11). The image shows successfulinternalization of FA-3WJ-LNA-miR21-Alexa647 RNP into GBM30 cells andaccumulation in cytoplasm not much in nucleus. Since LNA-miR21 will workagainst mature miR-21 in cytoplasm to show its small RNA interferingactivity, the cytoplasmic distribution of FA-3WJ-LNA-miR21-Alexa647 RNPpromises the drugability in target therapy of glioblastoma. Alexa647 wasexpressed in red peudocolor. The cytoskeleton of the fixed cells wasstained by Alexa Fluor 488 Phalloidin (Invitrogen, Grand Island, N.Y.)and the nucleus stained with 0.01% DAPI solution. Fluorescencemicroscopy was performed using Olympus 4-filter-based FluoViewFV1000-Filter Confocal Microscope System (Olympus Corp.) at thewavelengths of 461 nm (for the cell nucleus stained by DAPI), 530 nm(for the cytoskeleton stained by Alexa Fluor 488 Phalloidin) and 665 nm(for the Alexa647). Images were analyzed by Olympus FluoView Viewersoftware ver. 4.0 (Olympus). The fluorescent images were obtained usingFluoView FV1000-Filter Confocal Microscope System (Olympus Corp.).

Example 5

This study shows anti-tumor effect of systemically deliveredFA-3WJ-LNA-miR21 RNP in human glioblastoma cells derived tumor in vivo(FIG. 12). For in vivo test, intracranial tumor in mice was induced byimplanting GBM-Luc cells expressing luciferase gene which enables thetracing of tumor size change. Bioluminescence signal measured from theresulted brain tumor is expected to correlate with tumor growth. Toestablish in vivo mouse model, GBM30-Luc cells—induced brain tumor wasprepared into two groups of mice (n=5). At 14 days post-surgery, 1 mg/kgof mouse body weight of FA-3WJ-LNA-miR21 RNP (or FA-3WJ-LNA-SC asnegative control) was injected through the mouse tail vein in 100 μL ofPBS for total of five times. After each injection, mice were subjectedto bioluminescence whole body imaging to detect the endogenousluciferase expression level. Mice were injected with 75 mg/kg Luciferin(Perkin Elmer, Waltham, Mass.), and anesthetized. Bioluminescence fromthe anesthetized mice was detected by ZFOV-24 zoom lens-installed IVISLumina Series III Pre-clinical In Vivo Imaging System (Perkin Elmer,Waltham, Mass.). The luminescence intensity was expressed as AveragedRadiance [p/s/cm²;/sr]. When a group of brain tumor-bearing mice (n=5)were systemically injected with FA-3WJ-LNA-miR21 (1 mg/kg in 100 μL ofPBS) for five times over 10 days, the luciferase activity rapidlydecreased compared to the mice group injected with FA-3WJ-LNA-SC controlRNP, indicating the anti-tumor effect of FA-3WJ-LNA-miR21. as the tumorgrew indicating no effect of the control RNP on luciferase geneexpression. FIG. 12A shows representative in vivo MRI images for tumorvolume and bioluminescence intensity for luciferase activity from bothFA-3WJ-LNA-miR21 or FA-3WJ-LNA-SC after five injections. FIG. 12B showstumor volumes calculated from mean fluorescence intensity compared toscrambled control group after five injections, p=0.023 (n=5).

Example 6

This study shows the Knock-down of endogenous miR-21 in mouse tumor bysystemically delivered FA-3WJ-LNA-miR21 (FIG. 13). In this study,LNA-miR21 sequences conjugated to FA-3WJ-LNA-miR21 RNP is expected tosilence endogenous miR-21 in mouse tumor induced by GBM30 cells. Afterfive times of systemic administration of FA-3WJ-LNA-miR21 RNP, the tumorwas dissected out of mouse brain. Total RNA was extracted from the tumortissue with Trizol reagent according to the manufacture's protocol. Theexpression level of miR-21 was determined by TaqMan MicroRNA expressionReverse-transcription analysis kit. snoRNA U6 was used as normalizationinternal control. Non-tumor brains serve to show endogenous level ofmiR-21 in normal brain cells. GBM30 cells-induced tumor regions showedrelatively higher expression of miR-21 compared to non-tumor region.When the mouse tumors were systemically treated with FA-3WJ-LNA-miR21RNP, the level of miR-21 in the mouse tumors significantly decreased atleast more than two times than the mouse tumors injected with negativecontrol RNP, FA-3WJ-LNA-SC. It critically demonstrated the anti-miR-21silencing activity of FA-3WJ-LNA-miR21 RNP in vivo mouse models aftersystemic administration.

Example 7

This study shows the regulation of miR-21 by systemically deliveredFA-3WJ-LNA-miR21 induced apoptotic pathway through recovery of Ptenprotein expression (FIG. 14). In this study, Pten expression has beenreported to be down regulated in glioblastoma, and identified previouslyas a primary silencing target of over-expressed miR-21 in glioblastoma.Data in FIG. 14 successfully demonstrated the anti-miR-21 silencingeffect of to FA-3WJ-LNA-miR21 RNP. To evaluate the miR-21 silencingeffect in the down stream miR-21 targets, western blotting analysis wasperformed on total proteins extracted from mouse tumors aftersystemically injection of FA-3WJ-LNA-miR21 RNP. FIG. 14A refers toWestern blotting identified up-regulation of Pten protein expression inthe mouse tumor treated with to FA-3WJ-LNA-miR21 RNP. The increased Ptenexpression resulted suppression of Akt activity, a primary down streamtarget of Pten pathway, which activated apoptosis pathway. Evidently,the rescue of Pten expression resulted apoptosis in tumor cells to tumorregression as observed in FIG. 12. The image data in this study wasanalyzed by ImageJ software. Pten expression was increased at least morethan four times in the mouse tumors treated with to FA-3WJ-LNA-miR21 RNPcompared to those with to FA-3WJ-LNA-SC RNP (p=0.022, n=5).

Example 8

This study shows knock-down of endogenous miR-21 in mouse tumor bysystemically delivered FA-3WJ-LNA-miR21 improved overall survival ofbrain tumor-bearing mice (FIG. 15). In this study, Kaplan-Meyer survivalcurve was used to compare overall survival rates of two braintumor-bearing mice groups treated with to FA-3WJ-LNA-miR21 RNP andnegative control RNP (to FA-3WJ-LNA-SC) after total of five timesystemic administrations. As shown above data, apoptosis in mouse braintumor region activated by the systemically injected FA-3WJ-LNA-miR21 RNPsignificantly improved the survival rate (p=0.0023, n=5). Mediansurvival rate of the FA-3WJ-LNA-miR21 RNP treated mice group was 23days, while the mice group treated with FA-3WJ-LNA-SC RNP showed 19 daysof median survival rate.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

REFERENCES

-   1. Lesniak, M. S.; Brem, H. Targeted Therapy For Brain Tumours. Nat.    Rev. Drug Discov. 2004, 3, 499-508.-   2. Guo, P. The Emerging Field Of RNA Nanotechnology. Nat.    Nanotechnol 2010, 5, 833-842.-   3. Guo, P.; Haque, F.; Hallahan, B.; Reif, R.; Li, H. Uniqueness,    Advantages, Challenges, Solutions, And Perspectives In Therapeutics    Applying RNA Nanotechnology. Nucleic Acid Ther. 2012, 22, 226-245.-   4. Shu, Y.; Pi, F.; Sharma, A.; Rajabi, M.; Haque, F.; Shu, D.;    Leggas, M.; Evers, B. M.; Guo, P. Stable RNA Nanoparticles As    Potential New Generation Drugs For Cancer Therapy. Adv. Drug Deliv.    Rev. 2014, 66, 74-89.-   5. Guo, P.; Zhang, C.; Chen, C.; Garver, K.; Trottier, M. Inter-RNA    Interaction Of Phage Phi29 Prna To Form A Hexameric Complex For    Viral DNA Transportation. Mol. Cell 1998, 2, 149-155.-   6. Shu, D.; Moll, W. D.; Deng, Z.; Mao, C.; Guo, P. Bottom-Up    Assembly Of RNA Arrays And Superstructures As Potential Parts In    Nanotechnology. Nano Lett. 2004, 4, 1717-1723.-   7. Shu, Y.; Haque, F.; Shu, D.; Li, W.; Zhu, Z.; Kotb, M.;    Lyubchenko, Y.; Guo, P. Fabrication Of 14 Different RNA    Nanoparticles For Specific Tumor Targeting Without Accumulation In    Normal Organs. RNA 2013, 19, 767-777.-   8. Shu, D.; Khisamutdinov, E. F.; Zhang, L.; Guo, P. Programmable    Folding Of Fusion RNA In Vivo And In Vitro Driven By Prna 3WJ Motif    Of Phi29 DNA Packaging Motor. Nucleic Acids Res. 2014, 42, E10.-   9. Jasinski, D. L.; Khisamutdinov, E. F.; Lyubchenko, Y. L.; Guo, P.    Physicochemically Tunable Polyfunctionalized RNA Square Architecture    With Fluorogenic And Ribozymatic Properties. ACS Nano 2014, 8,    7620-7629.-   10. Khisamutdinov, E. F.; Li, H.; Jasinski, D. L.; Chen, J.; Fu, J.;    Guo, P. Enhancing Immunomodulation On Innate Immunity By Shape    Transition Among RNA Triangle, Square And Pentagon Nanovehicles.    Nucleic Acids Res. 2014, 42, 9996-10004.-   11. Khisamutdinov, E. F.; Jasinski, D. L.; Guo, P. RNA As A    Boiling-Resistant Anionic Polymer Material To Build Robust    Structures With Defined Shape And Stoichiometry. ACS Nano 2014.-   12. Shu, D.; Shu, Y.; Haque, F.; Abdelmawla, S.; Guo, P.    Thermodynamically Stable RNA Three-Way Junction For Constructing    Multifunctional Nanoparticles For Delivery Of Therapeutics. Nat.    Nanotechnol 2011, 6, 658-667.-   13. Haque, F.; Shu, D.; Shu, Y.; Shlyakhtenko, L. S.; Rychahou, P.    G.; Evers, B. M.; Guo, P. Ultrastable Synergistic Tetravalent RNA    Nanoparticles For Targeting To Cancers. Nano Today 2012, 7, 245-257.-   14. Abdelmawla, S.; Guo, S.; Zhang, L.; Pulukuri, S. M.; Patankar,    P.; Conley, P.; Trebley, J.; Guo, P.; Li, Q. X. Pharmacological    Characterization Of Chemically Synthesized Monomeric Phi29 Prna    Nanoparticles For Systemic Delivery. Mol. Ther. 2011, 19, 1312-1322.-   15. Rush, D. Periconceptional Folate And Neural Tube Defect. Am. J.    Clin. Nutr. 1994, 59, 511S-515S; Discussion 515S-516S.-   16. Grapp, M.; Just, I. A.; Linnankivi, T.; Wolf, P.; Lucke, T.;    Hausler, M.; Gartner, J.; Steinfeld, R. Molecular Characterization    Of Folate Receptor 1 Mutations Delineates Cerebral Folate Transport    Deficiency. Brain 2012, 135, 2022-2031.-   17. Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.; Frasca,    V.; Zurawski, V. R., Jr; Kamen, B. A. Distribution Of The Folate    Receptor GP38 In Normal And Malignant Cell Lines And Tissues. Cancer    Res. 1992, 52, 3396-3401.-   18. Steinfeld, R.; Grapp, M.; Kraetzner, R.; Dreha-Kulaczewski, S.;    Helms, G.; Dechent, P.; Wevers, R.; Grosso, S.; Gartner, J. Folate    Receptor Alpha Defect Causes Cerebral Folate Transport Deficiency: A    Treatable Neurodegenerative Disorder Associated With Disturbed    Myelin Metabolism. Am. J. Hum. Genet. 2009, 85, 354-363.-   19. Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.;    Leamon, C. P. Folate Receptor Expression In Carcinomas And Normal    Tissues Determined By A Quantitative Radioligand Binding Assay.    Anal. Biochem. 2005, 338, 284-293.-   20. Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Discovery And    Development Of Folic-Acid-Based Receptor Targeting For Imaging And    Therapy Of Cancer And Inflammatory Diseases. Acc. Chem. Res. 2008,    41, 120-129.-   21. Croce, C. M. Causes And Consequences Of Microrna Dysregulation    In Cancer. Nat. Rev. Genet. 2009, 10, 704-714.-   22. Shu, Y.; Shu, D.; Haque, F.; Guo, P. Fabrication Of Prna    Nanoparticles To Deliver Therapeutic Rnas And Bioactive Compounds    Into Tumor Cells. Nat. Protoc. 2013, 8, 1635-1659.-   23. Cheng, L.; Bao, S.; Rich, J. N. Potential Therapeutic    Implications Of Cancer Stem Cells In Glioblastoma. Biochem.    Pharmacol. 2010, 80, 654-665.-   24. Mao, P.; Joshi, K.; Li, J.; Kim, S. H.; Li, P.; Santana-Santos,    L.; Luthra, S.; Chandran, U. R.; Benos, P. V.; Smith, L.; et al.    Mesenchymal Glioma Stem Cells Are Maintained By Activated Glycolytic    Metabolism Involving Aldehyde Dehydrogenase 1A3. Proc. Natl. Acad.    Sci. U.S.A. 2013, 110, 8644-8649.-   25. Peruzzi, P.; Bronisz, A.; Nowicki, M. O.; Wang, Y.; Ogawa, D.;    Price, R.; Nakano, I.; Kwon, C. H.; Hayes, J.; Lawler, S. E. et al.    Microrna-128 Coordinately Targets Polycomb Repressor Complexes In    Glioma Stem Cells. Neuro Oncol. 2013, 15, 1212-1224.-   26. Li, J.; Zhu, S.; Kozono, D.; Ng, K.; Futalan, D.; Shen, Y.;    Akers, J. C.; Steed, T.; Kushwaha, D.; Schlabach, M. et al.    Genome-Wide Shrna Screen Revealed Integrated Mitogenic Signaling    Between Dopamine Receptor D2 (DRD2) And Epidermal Growth Factor    Receptor (EGFR) In Glioblastoma. Oncotarget 2014, 5, 882-893.-   27. Martin-Villalba, A.; Okuducu, A. F.; Von Deimling, A. The    Evolution Of Our Understanding On Glioma. Brain Pathol. 2008, 18,    455-463.-   28. Leamon, C. P.; Parker, M. A.; Vlahov, I. R.; Xu, L. C.;    Reddy, J. A.; Vetzel, M.; Douglas, N. Synthesis And Biological    Evaluation Of EC20: A New Folate-Derived, (99m)Tc-Based    Radiopharmaceutical. Bioconjug. Chem. 2002, 13, 1200-1210.-   29. Ciafre, S. A.; Galardi, S.; Mangiola, A.; Ferracin, M.; Liu, C.    G.; Sabatino, G.; Negrini, M.; Maira, G.; Croce, C. M.;    Farace, M. G. Extensive Modulation Of A Set Of Micrornas In Primary    Glioblastoma. Biochem. Biophys. Res. Commun. 2005, 334, 1351-1358.-   30. Suh, S. S.; Yoo, J. Y.; Nuovo, G. J.; Jeon, Y. J.; Kim, S.;    Lee, T. J.; Kim, T.; Bakacs, A.; Alder, H.; Kaur, B. et al.    Micrornas/TP53 Feedback Circuitry In Glioblastoma Multiforme. Proc.    Natl. Acad. Sci. U.S.A. 2012, 109, 5316-5321.-   31. Quintavalle, C.; Garofalo, M.; Zanca, C.; Romano, G.; Iaboni,    M.; Del Basso De Caro, M.; Martinez-Montero, J. C.; Incoronato, M.;    Nuovo, G.; Croce, C. M. et al. Mir-221/222 Overexpession In Human    Glioblastoma Increases Invasiveness By Targeting The Protein    Phosphate Ptpmu. Oncogene 2012, 31, 858-868.-   32. Quintavalle, C.; Donnarumma, E.; Iaboni, M.; Roscigno, G.;    Garofalo, M.; Romano, G.; Fiore, D.; De Marinis, P.; Croce, C. M.;    Condorelli, G. Effect Of Mir-21 And Mir-30b/C On TRAIL-Induced    Apoptosis In Glioma Cells. Oncogene 2013, 32, 4001-4008.-   33. Yoo, J. Y.; Pradarelli, J.; Haseley, A.; Wojton, J.; Kaka, A.;    Bratasz, A.; Alvarez-Breckenridge, C. A.; Yu, J. G.; Powell, K.;    Mazar, A. P. et al. Copper Chelation Enhances Antitumor Efficacy And    Systemic Delivery Of Oncolytic HSV. Clin. Cancer Res. 2012, 18,    4931-4941.

What is claimed is:
 1. A method of treating a brain tumor in a subjecthaving or at risk of developing a brain tumor, the method comprisingadministering to the subject a therapeutically effective amount of acomposition comprising an artificial RNA nanostructure molecule, whereinthe molecule comprises a multiple branched RNA junction motif comprisingat least one RNA oligonucleotide, and a brain tumor targeting module,wherein the module is coupled to an RNA junction motif, wherein themultiple branched RNA comprises a nucleotide sequence 5′-UUG CCA UGU GUAUGU GGG AUC CCG CGG CCA UGG CGG CCG GGA G-3′ (SEQ ID NO: 6) or5′-GATAAGCT CTC CCG GCC GCC ATG GCC GCG GGA T-3′ (SEQ ID NO: 7).
 2. Amethod of preventing brain tumor recurrence in a subject having or atrisk of having brain tumor recurrence, the method comprisingadministering to the subject a therapeutically effective amount of acomposition comprising an artificial RNA nanostructure molecule, whereinthe molecule comprises a multiple branched RNA junction motif comprisingat least one RNA oligonucleotide, and a brain tumor targeting module,wherein the module is coupled to an RNA junction motif wherein themultiple branched RNA comprises a nucleotide sequence 5′-UUG CCA UGU GUAUGU GGG AUC CCG CGG CCA UGG CGG CCG GGA G-3′ (SEQ ID NO: 6) or5′-GATAAGCT CTC CCG GCC GCC ATG GCC GCG GGA T-3′ (SEQ ID NO: 7).
 3. Themethod of claim 1, wherein the composition further comprises apharmaceutically acceptable carrier.
 4. The method of claim 1, whereinthe subject is a mammal or a non-mammal vertebrate.
 5. The method ofclaim 1, wherein the subject is a human.
 6. The method of claim 1,wherein the brain tumor is glioblastoma.
 7. The method of claim 1,wherein the molecule further comprises at least one bioactive agentcoupled to the RNA junction motif.
 8. The method of claim 1, wherein theRNA oligonucleotide comprises at least one chemical modification at the2′ position.
 9. The method of claim 8, wherein the modificationcomprises 2′ Fluoro, 2′ Amine, 2′ O-Methyl, or a combination thereof.10. The method of claim 1, wherein the motif is a three-branched RNAjunction motif.
 11. The method of claim 1, wherein the diameter of themolecule is at least about 40 nm or less.
 12. The method of claim 1,wherein the molecule has a zeta potential ranging from about −50 m V toabout 50 m V.
 13. The method of claim 10, wherein a branch of thethree-branched RNA junction motif comprises an a3WJ RNA module (SEQ IDNO: 1); a b3WJ RNA module (SEQ ID NO: 2); a c3WJ RNA module (SEQ ID NO:3); or a combination thereof.
 14. The method of claim 1, wherein RNAoligonucleotides comprises at least 6 nucleotides in length.
 15. Themethod of claim 1, wherein the brain tumor targeting module comprises aligand that binds to at least one brain tumor cell surface marker. 16.The method of claim 15, wherein the ligand binds to a folate receptor,an EGFR, a transferrin receptor, an RGD, or a combination thereof. 17.The method of claim 15, wherein the ligand comprises an aptamer.
 18. Themethod of claim 17, wherein the aptamer binds to EGFR, PDGFR, folatereceptor, or a combination thereof.
 19. The method of claim 1, whereinthe targeting module comprises a folate.
 20. The method of claim 7,wherein the bioactive agent comprises a drug, a therapeutic agent, afluorescent dye, a chemical, an siRNA, an miRNA, an anti-miRNA, aribozyme RNA, an antisense RNA or a combination thereof.
 21. The methodof claim 7, wherein the bioactive agent is directed to a brain tumormarker.
 22. The method of claim 20, the microRNA sequence is at least 6nucleotide in length.
 23. The method of claim 20, wherein the bioactiveagent is an anti-miRNA molecule for a miRNA comprising miR-9, miR-10b,miR-21, miR-17, or miR-26.
 24. The method of claim 20, wherein thebioactive agent is a miRNA molecule for a miRNA comprising let-7a,miR-10b, miR-25, miR-34a, miR-124, miR-145, or miR-181b.
 25. The methodof claim 23, wherein the anti-miRNA comprises an anti-miRNA lockednucleic acid (LNA) molecule.
 26. The method of claim 23, wherein theanti-miRNA LNA molecule comprises sequence 5′-GATAAGCT-3′,5′-AGCACTTT-3′, or 5′-ATTTGCAC-3′.
 27. The method of claim 20, whereinthe siRNA binds to an mRNA molecule encodes a protein comprising VEGF,EGFR, POK, AKT, AGT, RAF, RAS, MAPK, ERK, MGMT, MMP-2, MMP-9, PDGF,PDGFR, IGF-1, HGF, mTOR, Cox-2 or TGFβ1.
 28. The method of claim 20,wherein the siRNA binds to a mRNA molecule that encodes RAS, cMET, HER2,MDM2, PIK3CA, AKT, CDK4, or a combination thereof.